Methods for Antimicrobial Susceptibility Testing

ABSTRACT

A method for determining the susceptibility of bacteria in a clinical sample comprising urine or an inoculant derived therefrom to an antibiotic agent may include the steps of a) inoculating a test portion of the clinical sample in a medium containing a predetermined concentration of the antibiotic agent; b) inoculating a control portion of the clinical sample in a medium that does not contain the antibiotic agent; c) incubating the test portion for an incubation period; d) incubating the control portion for the incubation period; e) determining a quantity of RNA in the test portion and a quantity of RNA in the control portion at the conclusion of the incubation period that is less than 480 minutes after the completion of step a); and f) determining a susceptibility of the bacteria to the antibiotic agent by comparing the quantity of RNA in the test portion to the quantity of the RNA in the control portion.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit under 35 U.S.C. § 119(e) ofU.S. provisional patent application Ser. No. 62/547,361, filed Aug. 18,2017 and entitled Methods For Antimicrobial Susceptibility Testing; U.S.provisional patent application Ser. No. 62/671,380, filed May 14, 2018and entitled Methods For Estimating Bacterial Density In Specimens ByMeasurement Of Ribosomal RNA; U.S. provisional patent application Ser.No. 62/552,332, filed Aug. 30, 2017 and entitled Device for Optimizationof Microorganism Growth in Liquid Culture; and PCT Application No.PCT/US18/45211, filed Aug. 3, 2018 and entitled Methods for Lysis ofCells Within a Sample. The contents of these applications beingincorporated herein in their entirety by reference.

FIELD

In one of its aspects, the present invention relates to a method ofdetermining the susceptibility of a microorganism to an antimicrobialagent, and more particularly to a method of determining of thesusceptibility of a microorganism to an antimicrobial agent thatcombines a molecular measure of susceptibility with a predeterminedconcentration of antimicrobial agent.

BACKGROUND

The analysis of biological fluid samples, particularly the detection ofcertain target molecules within a biological fluid, has many clinicalapplications. For example, the isolation and identification ofuropathogens in urine samples is an important aspect of the clinicalmanagement of patients with urinary tract infections (UTIs) and otherinfectious diseases.

Culture-based methods for isolating and identifying uropathogens areknown in the art; however, these methods can be time consuming, laborintensive, and are not cost effective. Recent advances in technologyhave allowed for the development of electrochemical DNA biosensors withmolecular diagnostic capabilities, including bacterial pathogendetection. To run a successful electrochemical assay, a target cell canfirst be lysed such that a nucleic acid molecule, such as RNA, can bereleased from within the cell. Thus, the use of electrochemical DNAbiosensors relies on the efficient lysis and release of target moleculesfrom the cells to be diagnosed. These cells may include, among others,prokaryotic cells such as Gram-negative bacteria or Gram-positivebacteria, or fungal cells, such as yeast.

In some circumstances, a biological fluid may contain microorganisms,such as bacteria, and it may be desirable to determine if a givenmicroorganism is susceptible to treatment by one or more antimicrobialagents. For example, if a biological fluid contains bacteria, it may beuseful to determine if the particular bacteria in the sample issusceptible to, or alternatively, is resistant to, one or moreantibiotics. The effectiveness of an antibiotic can vary with theresistance of a bacterial pathogen to the antibiotic. Therefore,determining the antimicrobial sensitivity of bacterial pathogens in aclinical specimen is a key step in the diagnosis and treatment ofinfectious diseases.

Two common methods of phenotypic antimicrobial susceptibility testing(“AST”) are broth microdilution and Kirby-Bauer disc diffusion. Whilesuch methods can be relatively accurate in determining the antimicrobialsensitivity of bacterial pathogens in clinical specimen, both arerelatively slow, requiring lengthy incubation times of the sample withthe antibiotics (up to 24 hours). Such methods also often require alengthy pre-incubation culturing period (24-72 hours) to generate theAST sample, can be relatively labor-intensive, and can be challenging toautomate.

Due to the relatively serious nature of infectious diseases, it can bethe case that treatment should not be delayed. Therefore, antibiotictreatment is frequently started before AST results can be obtained usingconventional, non-molecular, and slow-acting testing methods. This canlead to a patient being given antibiotics, or other antimicrobialagents, without first knowing if the particular bacteria afflicting thepatient is susceptible or resistant to the particular antibioticadministered. If the bacteria are in fact resistant, the initial courseof antibiotics may be ineffective, which may contribute to a knownproblem/trend of patients receiving unnecessary or less effectiveantibiotics when other, potentially more effective antibiotics may havebeen available for use. This can be particularly problematic due to therise in drug-resistant microorganisms.

Despite the advances made to date in determining the antimicrobialsensitivity of bacterial pathogens in a clinical specimen, there is roomfor improvement to address the above-mentioned problems and shortcomingsof the prior art.

SUMMARY

It is an object of the present invention to obviate or mitigate at leastone of the above-mentioned disadvantages of the prior art.

It is another object of the present invention to provide a novel methodfor determining the susceptibility of a microorganism to anantimicrobial agent.

Accordingly, in one of its aspects, the present invention provides amethod for determining the susceptibility of a bacteria in a clinicalsample comprising urine or an inoculant derived therefrom to anantibiotic agent, the method comprising: (a) inoculating a test portionof a clinical sample in a medium containing a predeterminedconcentration of an antibiotic agent; (b) inoculating a control portionof the urine sample in a medium that does not contain the antibioticagent; (c) incubating the test portion for an incubation period; (d)incubating the control portion for the incubation period; (e)determining a quantity of RNA in the test portion and quantity of RNA inthe control portion at the conclusion of the incubation period that isless than 420 minutes after the completion of step a); and (f)determining a susceptibility of the bacteria to the antibiotic agent bycomparing the quantity of RNA in the test portion to the quantity of theRNA in the control portion.

In another of its aspects, the present invention provides a method ofdetermining the susceptibility of a microorganism in a sample comprisinga bodily fluid or an inoculant derived therefrom to at least twodifferent antimicrobial agents, the method comprising the steps of: (a)inoculating a first test portion of the sample in a medium containing afirst predetermined concentration of a first antimicrobial agent; (b)inoculating a second test portion of the sample in a medium containing asecond a predetermined concentration of a second antimicrobial agent;(c) inoculating a control portion of the sample in a medium that doesnot contain either the first or second antimicrobial agents; (d)incubating the first test portion for a first incubation period, thesecond test portion for a second incubation period, and the controlportion for a control incubation period, wherein each of the firstincubation period, the second incubation period, and the controlincubation period are less than 420 minutes; (e) determining a quantityof a nucleic acid molecule in the first test portion at the conclusionof the first incubation period, determining a quantity of the nucleicacid molecule in the second test portion at the conclusion of the secondincubation period and determining a quantity of the nucleic acidmolecule in the control portion at the conclusion of the controlincubation period; (f) determining a susceptibility of the microorganismto the first antimicrobial agent by comparing the quantity of thenucleic acid molecule in the first test portion to the quantity of thequantity of the nucleic acid molecule in the control portion; and (g)determining a susceptibility of the microorganism to the secondantimicrobial agent by comparing the quantity of the nucleic acidmolecule in the second test portion to the quantity of the quantity ofthe nucleic acid molecule in the control portion.

In another of its aspects, the present invention provides a method fordetermining the susceptibility of a microorganism in a sample to anantimicrobial agent, the method comprising: (a) inoculating a testportion of the sample in a medium containing a predeterminedconcentration of an antimicrobial agent; (b) inoculating a controlportion of the sample in a medium that does not contain theantimicrobial agent; (c) incubating the test portion and the controlportion for an incubation period that is less than 420 minutes; (d)determining a quantity of a nucleic acid molecule in the test portionand quantity of the nucleic acid molecule in the control portion at theconclusion of the incubation; and (e) determining a susceptibility ofthe microorganism to the antimicrobial agent by comparing the quantityof the nucleic acid molecule in the test portion to the quantity of thequantity of the nucleic acid molecule in the control portion.

Thus, the present inventors have developed a novel method fordetermining the antimicrobial susceptibility of a microorganism in aclinical specimen. This method uses a molecular measure of thesusceptibility of a microorganism to a given antimicrobial agent using apre-determined, non-standard and concentration of the antimicrobialagent (as compared to the concentrations that would be used in other,non-molecular susceptibility testing procedures). When using a molecularmeasurement technique, the growth of a given microorganism during thetest process can be determined by measuring the presence, absence, orrelative concentrations of target molecular features as a proxy forgrowth, such as, in some of the examples described herein, nucleic acidmolecules within the microorganisms.

The methods described herein may include comparing the quantity of anucleic acid molecule from a microorganism that has not been exposed toan antimicrobial agent to the quantity of a nucleic acid molecule from amicroorganism that has been exposed to an enhanced concentration of anantimicrobial agent. This method may help facilitate for a fasterdistinction between antimicrobial susceptible and antimicrobialresistant populations of microorganisms in a clinical specimen, ascompared to the conventional AST methods.

Some methods of quantifying nucleic acid molecules in a sample, such asbacterial ribosomal RNA (“rRNA”), can generally include the steps of: 1)Lysis to release rRNA; 2) Neutralization; 3) Hybridization of targetrRNA with a capture probe and detector probe; and 4) Detection ofcapture probe—target rRNA—detector probe complexes.

The lysing operations may be conducted using suitable lysing techniques,including those described herein. Determination of rRNA concentrationmay be based on a linear log-log correlation between the assay signaland rRNA analyte concentration. A synthetic target molecule at a knownconcentration may be included as a positive control for normalization ofassay signal intensity, whereby the assay signal generated by a samplemay be compared with the positive control result to determine the numberof target rRNA molecules per volume tested (concentration).

It is generally known that the number of a given target nucleic acidmolecule, such as the number of rRNA copies, per cell may vary widelybetween specimens/microorganisms. For example, rRNA copies per cell incultivated specimens may vary from as high as approximately 100,000copies per cell to as low as approximately 6,000 copies per cell,depending on the growth phase and density of bacteria cultivated in thegrowth medium. It was previously believed that such variation may makeit difficult to satisfactorily determine a quantity of the microorganismbased on the significantly variable number of nucleic acid molecules ina test sample. Therefore, one aspect of the teachings herein is relatedto a novel method for estimating bacterial or microorganism density in aspecimen based on the quantity of a target nucleic acid molecule withinthe specimen.

As described herein, utilizing the molecular counting/quantificationtechniques described herein may help provide acceptably accurate resultsfrom an AST in a relatively faster time than can be achieved usingconventional visual and/or microscopic inspection quantificationtechniques when testing similar cellular material, under similarincubation conditions, and when utilizing a similar dosage/concentrationof an antimicrobial agent. However, the inventors have also discoveredthat the length of incubation time that is required for a given AST canbe modified by changing the concentration of the antimicrobial agentthat is used to a pre-determined concentration.

To the knowledge of the inventors, a method of determining theantimicrobial susceptibility of a microorganism having such acombination of features is heretofore unknown.

Other advantages of the teachings described herein may become apparentto those of skill in the art upon reviewing the present specification.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the present invention will be described with reference tothe accompanying drawings, wherein like reference numerals denote likeparts, and in which:

FIG. 1 depicts a graph comparing EC6210 growth in a 96-well plate,incubation disc in a shaker, and incubation disc in a new incubator byLuminex signal.

FIG. 2 depicts levels of microorganism in various samples after culturewith ampicillin for 60 minutes. RiboResponse % refers to the percentageof ribosomal RNA calculated in the culture with ampicillin compared tothe amount in a control lacking ampicillin.

FIG. 3 depicts levels of microorganism in various samples after culturewith ampicillin for 90 minutes. RiboResponse % refers to the percentageof ribosomal RNA calculated in the culture with ampicillin compared tothe amount in a control lacking ampicillin.

FIG. 4 depicts levels of microorganism in various samples after culturewith cefazolin for 60 minutes. RiboResponse % refers to the percentageof ribosomal RNA calculated in the culture with cefazolin compared tothe amount in a control lacking cefazolin.

FIG. 5 depicts levels of microorganism in various samples after culturewith cefazolin for 90 minutes. RiboResponse % refers to the percentageof ribosomal RNA calculated in the culture with cefazolin compared tothe amount in a control lacking cefazolin.

FIG. 6 depicts levels of microorganism in various samples after culturewith ceftriaxone for 90 minutes. RiboResponse % refers to the percentageof ribosomal RNA calculated in the culture with ceftriaxone compared tothe amount in a control lacking ceftriaxone.

FIG. 7 depicts levels of RiboResponse % over time of samples consideredto be either susceptible or resistance to ceftriaxone after exposure to32 μg/mL of ceftriaxone. RiboResponse % refers to the percentage ofribosomal RNA calculated in the culture with ceftriaxone compared to theamount in a control lacking ceftriaxone.

FIG. 8 illustrates copies of ribosomal RNA of a positive control (i.e.,no antibiotic exposure) over time. Overlaid on the positive control datais theoretical examples of copies of ribosomal RNA of resistant andsusceptible bacteria over time. As depicted, the curve of rRNA copiesfor resistant bacteria would be similar to that of the positive controlfor growth.

FIG. 9 is a preferred embodiment of an apparatus for use in carrying outmechanical lysis comprising a spin platform (left) and centrifugal disk(right);

FIG. 10 illustrates improved cell lysis using a combination ofmechanical lysis and non-mechanical lysis;

FIG. 11 illustrates improved cell lysis using a combination ofmechanical lysis and non-mechanical lysis for a broad variety ofGram-positive bacteria;

FIG. 12 illustrates optimal signal with a combination of mechanicallysis (OmniLyse®) plus NaOH for Gram-positive bacteria;

FIG. 13 illustrates improved signal with a combination of mechanicallysis (OmniLyse®) plus NaOH for a broad variety of Gram-positivebacteria;

FIG. 14 illustrates rRNA detection for various NaOH concentrations andmechanical lysis durations;

FIG. 15 illustrates Luminex signal after NaOH treatment from 0 to 5minutes following a 1-minute mechanical lysis (OmniLyse®).

FIG. 16 illustrates a comparison of different enzyme concentrations whenused in biological lysis of Gram-positive cells.

FIG. 17A illustrates a comparison of differing lengths of time ofmechanical lysis (OmniLyse®) in combination with alkaline lysis.

FIG. 17B illustrates a comparison of different concentrations of NaOH incombination with mechanical lysis (OmniLyse®).

FIG. 18 illustrates the Luminex signal after lysing certain types ofcells, including Gram-negative cells, Gram-positive cells, and yeastcells.

FIG. 19 illustrates the effect of different buffers used to neutralize acell lysate.

FIG. 20, in a flowchart, illustrates the steps involved in quantifyingbacterial density in a urine specimen using the rRNA concentration ofbacteria in the specimen;

FIG. 21, in a graph, illustrates the correlation between rRNAconcentration and density of E. coli in urine specimens from patientswith urinary tract infection;

FIG. 22, in a graph, illustrates the correlation between rRNA copies percell and density of E. coli in urine specimens from patients withurinary tract infection;

FIG. 23, in a graph, illustrates the contrast between rRNA copies percell and density of E. coli cultivated in growth medium vs. E. coli inurine specimens from patients with urinary tract infection; and

FIG. 24, in a graph, illustrates AST assay results for Ceftriaxone whenincubation was conducted on a centrifugal disc.

DETAILED DESCRIPTION

Various apparatuses or processes will be described below to provide anexample of an embodiment of each claimed invention. No embodimentdescribed below limits any claimed invention and any claimed inventionmay cover processes or apparatuses that differ from those describedbelow. The claimed inventions are not limited to apparatuses orprocesses having all of the features of any one apparatus or processdescribed below or to features common to multiple or all of theapparatuses described below. It is possible that an apparatus or processdescribed below is not an embodiment of any claimed invention. Anyinvention disclosed in an apparatus or process described below that isnot claimed in this document may be the subject matter of anotherprotective instrument, for example, a continuing patent application, andthe applicants, inventors, or owners do not intend to abandon, disclaim,or dedicate to the public any such invention by its disclosure in thisdocument.

Conventional methods for determining the bacterial (microbial) densityin a sample (whether as a standalone process or part of a multi-stageassay such as an AST) often require at least one growth phase, in whichan enriched bacterial culture is prepared from the specimen. Suchmethods may be relatively accurate but may tend to be relatively slow,taking several hours, days, or weeks to provide useful results. Inaddition to the time required for determining the microbial density,conventional AST methods for determining the susceptibility of amicroorganism to an antimicrobial agent in a sample may be relativelyaccurate but tend to be relatively slow, taking several hours or days tocomplete. In a clinical environment, such time frames may be undesirableand may be considered too long a time period to withhold/delay treatmentfor a subject. This time delay can sometimes lead to treatments beingimplemented, such as a particular antibiotic being prescribed before theAST results are obtained. This may lead to the unnecessary prescriptionof antibiotics and/or the prescription of an antibiotic that is lesseffective in treating a particular infection than other availableantibiotics. In some circumstances, time may be of the essence whendetermining the susceptibility of a bacteria, or other microorganism, toan antimicrobial agent.

For example, a given clinical specimen may be obtained from a subjectwith a suspected infection who may require further medical treatmentbased on the results of the analysis of the clinical specimen. Forexample, urine specimens are often obtained from subjects experiencingsymptoms consistent with urinary tract infections. In thesecircumstances, it may be desirable to analyze the specimen's response toa variety of different antibiotic agents that could possibly beprescribed to the subject and to determine which of such agents islikely to be relatively more or less effective than the others. Forconvenience, such analysis would preferably be conducted in a relativelyshort time period, such as during a routine doctor's visit or in aperiod of time that the subject might be reasonably expected to wait atthe testing location. Preferably, this time period may be less thanabout 4 hours (or other time limits mentioned herein), and morepreferably may be less than about 90 minutes or less than about 60minutes. This may help a clinician obtain the results while thesubject/patient waits, and to then prescribe a desired antibiotic agentfor treatment.

Optionally, a particular clinical sample may be tested with respect totwo or more antimicrobial agents simultaneously. For example, a clinicalsample may be sub-divided into two or more test portions, along with atleast one control portion, that can be separately, but simultaneouslytested. In some arrangements, a clinical sample may be sub-divided intoseven test portions and one control portion, with each test portionbeing exposed to a different antimicrobial agent during their respectiveincubation periods and then being evaluated with respect to a commoncontrol portion.

Preferably, tests that are being conducted in parallel may be configuredso that the respective incubation periods for each of the test portionsare approximately equal, whereby each of the test portions can beprocessed/quantified at about the same time. This may also helpfacilitate the use of a common control portion, as compared to operatingtests with different incubation periods which may preferably be comparedto different, respective control portions having substantially the sameincubation period. Configuring each of the test and control incubationportions to be about the same, such as each being about 90 minutes orabout 60 minutes, may help reduce the need for an operator or technicianto monitor the tests at different time intervals, and may allow anoperator to initiate all of the tests and then only need to return tocollect the results at the end of the pre-set incubation period (i.e.,set a machine to perform the tests and only have to return after 60 or90 minutes have passed, rather than having to return at different timesto observe the results of the different tests).

In some circumstances, the variety of different antimicrobial agents tobe tested may have incubation periods that are sufficiently similarunder the expected testing conditions and using conventionalconcentration/dosages. In other circumstances, utilizing conventionalconcentrations/dosages of the antimicrobial agents may lead toincubation times that are different, and do not lend themselves to beingprocessed/quantified at the same time and/or being compared to a commoncontrol portion. To help facilitate the parallel/simultaneous testing ofdifferent antimicrobials, the inventors have discovered that modifyingthe concentration/dosage of a given antimicrobial agent can affect thelength of its associated incubation period, under otherwise similarconditions. For example, the inventors have discovered, as describedherein, that a given antimicrobial can be provided in a predeterminedconcentration that can help provide an incubation period having atargeted length of time—such as about 90 minutes or about 60 minutes. Ithas also been discovered that the predetermined concentration that isused to provide an incubation period of about 90 minutes, for example,may be different for different antimicrobial agents. In such cases, eachantimicrobial agent may be provided in a different, predeterminedconcentration such that each of the tests to be conducted can each haveapproximately the same incubation period. In these examples, the targetparameter that is to be achieved is a desired incubation time that canbe synchronized with the incubation times for other tests beingconducted in parallel. In some other examples, instead of configuringthe incubation period to have a target duration/length, thepredetermined, concentration could be selected to target anotherparameter, such as configuring an AST to provide useful results in theshortest possible time frame, combined with the molecular analysistechniques, or to configuring an AST to provide useful results whileconsuming a relatively small amount of the particular antimicrobialagent (regardless of the incubation time), or a balance of all of thesefactors.

For example, a blood sample may be obtained from a patient experiencingsymptoms consistent sepsis or other bloodborne, microorganism-basedconditions. In such circumstances, completing a suitable accurate AST inthe shortest practical time may be desirable, even if the testing ofdifferent antimicrobial agents requires different incubation periods.Treatment for the patient could then begin once the first acceptableantimicrobial agent has been identified, rather than waiting until theend of the longest of the incubation periods. Such tests may be likelyto be performed in hospitals or other such environments, wheresufficient staff can be available to conduct and monitor a variety oftests in parallel. In other examples, an apparatus for conducting suchtests may be configured to automatically read the results from eachseparate test at different times. To help facilitate this approach,additional control portions can be used, and preferably, at least onecontrol portion can be provided for each test portion to be analyzed(i.e., pairs of corresponding test and control portions can beprovided). As each test portion reaches the end of its incubationperiod, it can be processed and compared to the condition of itsrespective control sample, as described herein. In these examples,predetermined concentration can be the concentration that provides theshortest incubation period without compromising the accuracy of the testresults. For a given antimicrobial agent, this may be different than thepredetermined concentration used when configuring the incubation periodto have a target duration.

In some other situations, it may be desirable to obtain useful testresults while minimizing the amount of the antimicrobial agent consumedduring the testing process. This may be desirable if the antimicrobialagent is in relatively short supply and/or is relatively expensive. Insuch examples, the predetermined concentration may be the minimal amountof a given antimicrobial agent that is sufficient to obtain useful, andacceptably accurate test results. This concentration may be differentthan the concentration in the other examples described herein.

In general, the performance and associated speed of performing themethods described herein can be related to techniques and methods usedfor the incubating, lysing, and quantifying the test specimens alongwith the predetermined concentration(s) of the antimicrobial agentsused. The particular predetermined, concentration for a givenantimicrobial to be used in a given circumstance (e.g. when trying toachieve a particular objective or effect on the incubation period) maybe selected based on the nature of the test being conducted, whether thetest is being conducted alone or in combination with the testing ofother antimicrobial agents, the urgency of the test results, and othersuch factors.

Preferably, an apparatus, such as a test cartridge or centrifugal disccan be pre-loaded with a predetermined, concentration of a givenantimicrobial and then made available to a clinic or user in acorresponding use circumstance. For example, an eight-channelcentrifugal disc can have one control channel and can have its otherchannels pre-loaded with seven different antimicrobial agents in,potentially different, predetermined concentrations so that all of thetest channels have an incubation period of about 60 minutes. Theparticular antimicrobial agents used can be pre-selected to be thosethat are available in a given region or that are, based on pastexperience, relatively likely to be effective against the types ofmicrobes that may be expected for a given test. For example, a UTIassessment disc could be pre-loaded with the seven antimicrobial agentsthat may be expected to be effective in treating the types of bacteriathat may be expected to be present in a clinical urine sample. Suchdiscs could be stocked in doctors' offices, clinics, and other suchlocations where patients may seek medical attention.

Furthermore, conventional quantification and AST techniques may requirea skilled technician to set-up and run the bacterial cultures, as wellas to interpret the results. The analysis may also require specializedand/or costly equipment. As such equipment and skilled technicians canbe relatively scarce resources, they are often located in centralizedlabs and/or hospital environments which are removed from commonfrontline care facilities, such as a physician's or veterinarian'soffice, walk-in clinics, and the like. This arrangement can furtherdelay the processing and analysis of clinical specimens by several hoursor days, as the specimens must be physically transported from thefront-line environment to a centralized testing location and may thenwait in a testing queue or backlog of samples awaiting analysis. Thistime-delay may reduce the accuracy of the ensuing clinical specimenanalysis due to such factors as growth or death of any bacteria that maybe present in the specimen.

Therefore, there remains a need for synchronizing the incubation timesfor different antimicrobial compounds to help perform multiple differenttests simultaneously, reducing the amount of a given antimicrobial agentrequired to obtain an accurate AST test result. There also remains aneed for relatively faster specimen analysis methods, and a need to beable to perform at least some of the analysis in situ in a front-linesetting, such as in a physician's or veterinarian's office, instead ofhaving to physically transport the specimens to a centralized location.Similarly, it would be advantageous to provide a method in which aclinically meaningful test result (i.e., information that can helpinform treatment decisions) can be provided to a caregiver withoutrequiring the individual skill and judgment of a skilled technician.

To help mitigate at least some of these deficiencies in conventionalmethods of specimen analysis, the present inventors have developed theprocess and methods described herein, including a method in which it maybe possible to estimate the microorganism density and susceptibility toan antimicrobial agent in a specimen in situ, in a front line setting,and in less time than conventional methods may allow for. In contrast tothe established practices of determining the susceptibility of amicroorganism to an antimicrobial agent, the present inventors havediscovered a method, which combines a molecular measure of antimicrobialsusceptibility with a predetermined concentration of antimicrobialagent, that may provide a faster distinction between antimicrobialsusceptible and antimicrobial resistant populations of microorganisms ina clinical specimen, as compared to conventional AST methods.

In addition to reducing the time required to perform AST on a clinicalspecimen, it may be desirable to determine the susceptibility of themicroorganisms in a specimen to multiple antimicrobial agents to ensuretreatment includes the most appropriate antibiotic or combination ofantibiotics. It may be further desirable to test such susceptibility tomultiple antimicrobials simultaneously/in parallel, thereby streamliningthe AST process by providing a single test in which the response tomultiple antimicrobials can be compared to a common control. As such,the present inventors have developed a method in which it may bepossible to estimate microorganism density and susceptibility tomultiple antimicrobial agents in a specimen in less time thanconventional methods may allow and utilizing a common incubation periodduration.

Disclosed herein are methods for determining the susceptibility of amicroorganism to one or more antimicrobial agents. Determining thesusceptibility of a microorganism to an antimicrobial agent may comprisecomparing the quantity of a nucleic acid molecule from a microorganismthat has not been exposed to an antimicrobial agent to the quantity of anucleic acid molecule from a microorganism that has been exposed to apredetermined concentration of an antimicrobial agent. Use of thepredetermined concentrations of antimicrobial agents in the methodsdisclosed herein may allow for faster antimicrobial susceptibilitytesting.

In accordance with one broad aspect of the teachings described herein, amethod for determining the susceptibility of bacteria in a clinicalsample comprising urine or an inoculant derived therefrom to anantibiotic agent, the method comprising: (a) inoculating a test portionof the clinical sample in a medium containing a predeterminedconcentration of the antibiotic agent; (b) inoculating a control portionof the clinical sample in a medium that does not contain the antibioticagent; (c) incubating the test portion for an incubation period; (d)incubating the control portion for the incubation period; (e)determining a quantity of RNA in the test portion and a quantity of RNAin the control portion at the conclusion of the incubation period thatis less than 420 minutes after the completion of step a); and (f)determining a susceptibility of the bacteria to the antibiotic agent bycomparing the quantity of RNA in the test portion to the quantity of theRNA in the control portion.

Preferred embodiments of this method may include any one or acombination of any two or more of any of the following features:

-   -   incubating the test portion is done within a test incubation        chamber on a centrifugal disc, and incubating the control        portion is done within a control incubation chamber on the same        centrifugal disc;    -   the test incubation chamber is fluidically isolated from the        control incubation chamber;    -   the RNA comprises pre-ribosomal RNA;    -   the RNA comprises mature RNA;    -   the RNA comprises ribosomal RNA;    -   the RNA comprises 16S rRNA;    -   the RNA comprises 23S rRNA;    -   the incubation period is equal to or less than 450 minutes;    -   the incubation period is equal to or less than 420 minutes;    -   the incubation period is equal to or less than 390 minutes;    -   the incubation period is equal to or less than 360 minutes;    -   the incubation period is equal to or less than 300 minutes;    -   the incubation period is equal to or less than 270 minutes;    -   the incubation period is equal to or less than 240 minutes;    -   the incubation period is equal to or less than 210 minutes;    -   the incubation period is equal to or less than 150 minutes;    -   the incubation period is equal to or less than 120 minutes;    -   the incubation period is equal to or less than 90 minutes;    -   the incubation period is equal to or less than 60 minutes;    -   the incubation period is equal to or less than 30 minutes;    -   the antibiotic agent is a bactericidal antibiotic;    -   the antibiotic agent is a bacteriostatic antibiotic;    -   the antibiotic agent comprises at least one of Gentamicin,        Ciprofloxacin, Cefazolin, Ceftriaxone, Cefepime, Ampicillin,        Trimethoprim-Sulfamethoxazole, Nitrofurantoin, Fosfomycin,        Amoxicillin-Clavulanate, Amikacin, Ertapenem, Meropenem and        combinations thereof;    -   the predetermined concentration is above the sensitive CLSI MIC        cutoff (for urine) for the antibiotic agent;    -   the predetermined concentration is above the intermediate CLSI        MIC cutoff (for urine) for the antibiotic agent;    -   the predetermined concentration is above the resistant CLSI MIC        cutoff (for urine) for the antibiotic agent;    -   the predetermined concentration is at least 2-fold or greater        than the resistant CLSI MIC cutoff (for urine) for the        antibiotic agent;    -   the predetermined concentration is at least 4-fold or greater        than the resistant CLSI MIC cutoff (for urine) for the        antibiotic agent;    -   the predetermined concentration is between the intermediate CLSI        MIC cutoff and the resistant CLSI MIC cutoff (for urine) for the        antibiotic agent;    -   the predetermined concentration is below the sensitive CLSI MIC        cutoff (for urine) for the antibiotic agent;    -   the sensitive CLSI MIC cutoff (for urine) is at least 2-fold or        greater than the predetermined concentration for the antibiotic        agent;    -   the antibiotic agent comprises Gentamicin and the predetermined        concentration is between about 2 μg/mL and 16 μg/mL;    -   the predetermined concentration is between about 2 μg/mL and 4        μg/mL;    -   the predetermined concentration is about 2 μg/mL;    -   the predetermined concentration is about 4 μg/mL;    -   the sensitive CLSI MIC cutoff (for urine) for Gentamicin is        equal to or greater than the predetermined concentration;    -   the antibiotic agent comprises Ciprofloxacin and the        predetermined concentration is between about 1 μg/mL and 8        μg/mL;    -   the predetermined concentration is between about 1 μg/mL and 4        μg/mL;    -   the predetermined concentration is about 4 μg/mL;    -   the predetermined concentration is substantially equal to the        resistant CLSI MIC cutoff (for urine) for Ciprofloxacin;    -   the antibiotic agent comprises Cefazolin and the predetermined        concentration is between about 2 μg/mL and about 256 μg/mL;    -   the predetermined concentration is between about 16 μg/mL and        about 128 μg/mL;    -   the predetermined concentration is about 64 μg/mL;    -   the predetermined concentration is substantially equal to 2        times the resistant CLSI MIC cutoff (for urine) for Cefazolin;    -   the antibiotic agent comprises Ceftriaxone and the predetermined        concentration is between about 1 μg/mL and about 128 μg/mL;    -   the predetermined concentration is between about 16 μg/mL and        about 64 μg/mL;    -   the predetermined concentration is about 32 μg/mL;    -   the predetermined concentration is substantially equal to 8        times the resistant CLSI MIC cutoff (for urine) for Ceftriaxone;    -   the antibiotic agent comprises Cefepime and the predetermined        concentration is between about 4 μg/mL and about 128 μg/mL;    -   the predetermined concentration is between about 16 μg/mL and        about 128 μg/mL;    -   the predetermined concentration is between about 32 μg/mL and        about 64 μg/mL;    -   the predetermined concentration is about 32 μg/mL;    -   the predetermined concentration is about 64 μg/mL;    -   the predetermined concentration is substantially equal to 2 or 4        times the resistant CLSI MIC cutoff (for urine) for Cefepime;    -   the antibiotic agent comprises Ampicillin and the predetermined        concentration is between about 8 μg/mL and about 2048 μg/mL;    -   the predetermined concentration is between about 128 μg/mL and        about 512 μg/mL;    -   the predetermined concentration is about 128 μg/mL;    -   the predetermined concentration is about 512 μg/mL;    -   the predetermined concentration is substantially equal to about        4 times the resistant CLSI MIC cutoff (for urine) for        Ampicillin;    -   the predetermined concentration is substantially equal to about        16 times the resistant CLSI MIC cutoff (for urine) for        Ampicillin;    -   the antibiotic agent comprises Trimethoprim-Sulfamethoxazole and        the predetermined concentration for Trimethoprim is between        about 2 μg/mL and about 16 μg/mL and the predetermined        concentration for Sulfamethoxazole is between about 38 μg/mL and        about 304 μg/mL;    -   the predetermined concentration for Trimethoprim is between        about 4 μg/mL and about 8 μg/mL and the predetermined        concentration for Sulfamethoxazole is between about 76 μg/mL and        about 152 μg/mL;    -   the predetermined concentration for Trimethoprim is about 4        μg/mL and the predetermined concentration for Sulfamethoxazole        is about 76 μg/mL;    -   the predetermined concentration for        Trimethoprim-Sulfamethoxazole is substantially equal to the        resistant CLSI MIC cutoff (for urine) for        Trimethoprim-Sulfamethoxazole;    -   the antibiotic agent comprises Nitrofurantoin and the        predetermined concentration is between about 4 μg/mL and about        512 μg/mL;    -   the predetermined concentration is between about 8 μg/mL and        about 32 μg/mL;    -   the predetermined concentration is about 16 μg/mL;    -   the sensitive CLSI MIC cutoff (for urine) for Nitrofurantoin is        at least 2-fold or greater than the predetermined concentration;    -   the antibiotic agent comprises Fosfomycin and the predetermined        concentration is between about 4 μg/mL and about 512 μg/mL;    -   the predetermined concentration is between about 8 μg/mL and        about 128 μg/mL;    -   the predetermined concentration is about 64 μg/mL;    -   the sensitive CLSI MIC cutoff (for urine) for Fosfomycin is at        about equal to the predetermined concentration;    -   the antibiotic agent comprises Amoxicillin-Clavulanate and the        predetermined concentration for Amoxicillin is between about 2        μg/mL and about 256 μg/mL and the predetermined concentration        for Clavulanate is between about 1 μg/mL and about 128 μg/mL;    -   the predetermined concentration for Amoxicillin is between about        8 μg/mL and about 128 μg/mL and the predetermined concentration        for Clavulanate is between about 4 μg/mL and about 64 μg/mL;    -   the predetermined concentration for Amoxicillin is about 64        μg/mL and the predetermined concentration for Clavulanate is        about 32 μg/mL;    -   the predetermined concentration for Amoxicillin is about 32        μg/mL and the predetermined concentration for Clavulanate is        about 16 μg/mL;    -   the predetermined concentration for Amoxicillin is about 16        μg/mL and the predetermined concentration for Clavulanate is        about 8 μg/mL;    -   the predetermined concentration is equal to the intermediate        CLSI MIC cutoff (for urine) for Amoxicillin-Clavulanate;    -   the predetermined concentration is greater than the intermediate        CLSI MIC cutoff (for urine) for Amoxicillin-Clavulanate;    -   the predetermined concentration is equal to or greater than the        resistant CLSI MIC cutoff (for urine) for        Amoxicillin-Clavulanate;    -   the antibiotic agent comprises Amikacin and the predetermined        concentration is between about 2 μg/mL and about 64 μg/mL;    -   the predetermined concentration is between about 8 μg/mL and        about 64 μg/mL;    -   the predetermined concentration is about 32 μg/mL;    -   wherein the predetermined concentration is about 16 μg/mL;    -   the predetermined concentration is about 8 μg/mL;    -   the predetermined concentration is less than the resistant CLSI        MIC cutoff (for urine) for Amikacin;    -   the predetermined concentration is equal to the intermediate        CLSI MIC cutoff (for urine) for Amikacin;    -   the predetermined concentration is less than or equal to the        sensitive CLSI MIC cutoff (for urine) for Amikacin;    -   the antibiotic agent comprises Ertapenem and the predetermined        concentration is between about 0.5 μg/mL and about 8 μg/mL;    -   the predetermined concentration is between about 1 μg/mL and        about 4 μg/mL;    -   the predetermined concentration is about 4 μg/mL;    -   the predetermined concentration is about 2 μg/mL;    -   the predetermined concentration is greater than or equal to the        resistant CLSI MIC cutoff (for urine) for Ertapenem;    -   the antibiotic agent comprises Meropenem and the predetermined        concentration is between about 1 μg/mL and about 8 μg/mL;    -   the predetermined concentration is between about 1 μg/mL and        about 4 μg/mL;    -   the predetermined concentration is about 4 μg/mL;    -   the predetermined concentration is about 2 μg/mL;    -   the predetermined concentration is equal to the resistant CLSI        MIC cutoff (for urine) for Meropenem;    -   the predetermined concentration is equal to the intermediate        CLSI MIC cutoff (for urine) for Meropenem;    -   determining a baseline quantity of RNA in the control portion        before the incubation period is complete and comparing the        baseline quantity of RNA to the quantity of RNA in the control        portion at the end of the incubation period to determine if the        quantity of RNA in the control portion increased by a        measurement threshold amount during the incubation period;    -   the bacteria comprises a Gram-negative bacterium;    -   the bacteria comprises a Gram-positive bacteria;    -   the bacteria is an unknown bacteria when steps a) to f) are        conducted;    -   lysing the test portion prior to determining the quantity of RNA        in the test portion;    -   further comprising the steps of:        -   g) subjecting the test portion to mechanical lysis to cause            disruption of a cellular membrane in the bacteria;        -   h) contacting the test portion with an alkaline material to            produce a lysate composition comprising the RNA; and        -   i) recovering the lysate composition from the test portion;    -   Step h) comprises contacting the bacteria in the test portion        with an alkaline liquid;    -   Step h) comprises contacting the bacteria in the test portion        with an alkaline solution;    -   the alkaline solution is a sodium hydroxide solution;    -   the alkaline solution has a concentration of 10M or less;    -   the alkaline solution has a concentration in the range of from        1M to 5M;    -   the alkaline solution has a concentration in the range of from        1.5M to 3M;    -   the alkaline solution has a concentration of 2M;    -   the alkaline solution has a concentration of 3M;    -   lysing the test portion comprises transferring an aliquot of an        inoculate to a lysing container;    -   incubating the test portion is done within a test incubation        chamber on a centrifugal disc, and lysing the test portion is        conducted within a lysing chamber on the same centrifugal disc;    -   the lysing chamber is fluidically connected to the test        incubation chamber;    -   the lysing chamber comprises the test incubation chamber;    -   Steps g) and h) are conducted for a period of 10 minutes or        less;    -   Steps g) and h) are conducted for a period of from 30 seconds to        10 minutes;    -   Steps g) and h) are conducted for a period of from 1 minute to 8        minutes;    -   Steps g) and h) are conducted for a period of from 2 minutes±30        seconds;    -   Steps g) and h) are conducted for a period of from 3 minutes±30        seconds;    -   Steps g) and h) are conducted for a period of from 4 minutes±30        seconds;    -   Steps g) and h) are conducted for a period of from 5 minutes±30        seconds;    -   Steps g) and h) are conducted for a period of from 6 minutes±30        seconds;    -   Steps g) and h) are conducted for a period of from 7 minutes±30        seconds;    -   Steps g) and h) are carried out concurrently;    -   the mechanical lysis comprises a combination of centrifugation        and puck lysing;    -   the mechanical lysis comprises a combination of centrifugation        and magnetic puck lysing;    -   the combination of centrifugation and puck lysing is carried out        in a common lysis chamber;    -   Steps h) and i) are carried out concurrently;    -   Steps h) and i) are carried out sequentially;    -   Step i) is carried out after commencement of disruption of the        cellular membrane in Step h);    -   the bacteria are susceptible to the antibiotic agent if the        quantity of RNA in the control portion is more than the quantity        of RNA in the test portion at the conclusion of the incubation        period;    -   the bacteria are not susceptible to the antibiotic agent if the        quantity of RNA in the control portion is nearly equal, equal,        or less than the quantity of RNA in the test portion at the        conclusion of the incubation period;    -   the microorganism is susceptible to the antibiotic agent when        the quantity of RNA in the test portion is about 40% or less of        the quantity of RNA in the control portion at the conclusion of        the incubation period; and    -   the microorganism is resistant to the antibiotic agent when the        quantity of RNA in the test portion is about 60% or more of the        quantity of RNA in the control portion at the conclusion of the        incubation period.

In another of its aspects, the present invention relates to a method ofdetermining the susceptibility of a microorganism in a sample comprisinga bodily fluid or an inoculant derived therefrom to at least twodifferent antimicrobial agents, the method comprising the steps of: (a)inoculating a first test portion of the sample in a medium containing afirst predetermined concentration of a first antimicrobial agent; (b)inoculating a second test portion of the sample in a medium containing asecond a predetermined concentration of a second antimicrobial agent;(c) inoculating a control portion of the sample in a medium that doesnot contain either the first or second antimicrobial agents; (d)incubating the first test portion for a first incubation period, thesecond test portion for a second incubation period, and the controlportion for a control incubation period, wherein each of the firstincubation period, the second incubation period, and the controlincubation period are less than 420 minutes; (e) determining a quantityof a nucleic acid molecule in the first test portion at the conclusionof the first incubation period, determining a quantity of a nucleic acidmolecule in the second test portion at the conclusion of the secondincubation period and determining a quantity of a nucleic acid moleculein the control portion at the conclusion of the incubation period; (f)determining a susceptibility of the microorganism to the firstantimicrobial agent by comparing the quantity of the nucleic acidmolecule in the first test portion to the quantity of the nucleic acidmolecule in the control portion; and (g) determining a susceptibility ofthe microorganism to the second antimicrobial agent by comparing thequantity of the nucleic acid molecule in the second test portion to thequantity of the nucleic acid molecule in the control portion.

Preferred embodiments of this method may include any one or acombination of any two or more of any of the following features:

-   -   the first incubation period is the same as the second incubation        period;    -   at least one of the first incubation period and the second        incubation period is the same as the control incubation period;    -   at least one of the first incubation period and the second        incubation period is less than the control incubation period    -   the first predetermined concentration and the second        predetermined concentration are different and are configured so        that the steps of determining the quantity of the nucleic acid        molecule in the first test portion at the conclusion of the        first incubation period and determining the quantity of the        nucleic acid molecule in the second test portion are performable        simultaneously;    -   the first incubation period is equal to or less than 420        minutes;    -   the first incubation period is equal to or less than 390        minutes;    -   the first incubation period is equal to or less than 360        minutes;    -   the first incubation period is equal to or less than 300        minutes;    -   the first incubation period is equal to or less than 270        minutes;    -   the first incubation period is equal to or less than 240        minutes;    -   the first incubation period is equal to or less than 210        minutes;    -   the first incubation period is equal to or less than 150        minutes;    -   the first incubation period is equal to or less than 120        minutes;    -   the first incubation period is equal to or less than 90 minutes;    -   the first predetermined concentration and the second        predetermined concentration are different and are configured so        that the first incubation period and the second incubation        period are substantially the same and are both equal to or less        than 90 minutes;    -   the first predetermined concentration and the second        predetermined concentration are different and are configured so        that the first incubation period and the second incubation        period are substantially the same and are both equal to or less        than 120 minutes;    -   the first incubation period is equal to or less than 60 minutes;    -   the first predetermined concentration and second predetermined        concentration are different and are configured so that the first        incubation period and the second incubation period are        substantially the same and are both equal to or less than 60        minutes;    -   the first incubation period is equal to or less than 30 minutes;    -   when the first predetermined concentration and second        predetermined concentration are the same but the first        incubation period and the second incubation period are        different;    -   the first predetermined concentration is different than the        second predetermined concentration;    -   the first antimicrobial agent comprises a first antibiotic agent        and the second antimicrobial agent comprises a second antibiotic        agent;    -   the antibiotic agent is a bactericidal antibiotic;    -   the antibiotic agent is a bacteriostatic antibiotic;    -   the antibiotic agent comprises at least one of Gentamicin,        Ciprofloxacin, Cefazolin, Ceftriaxone, Cefepime, Ampicillin,        Trimethoprim-Sulfamethoxazole, Nitrofurantoin, Fosfomycin,        Amoxicillin-Clavulanate, Amikacin, Ertapenem, Meropenem and        combinations thereof;    -   the predetermined concentration is above the sensitive CLSI MIC        cutoff (for urine) for the antibiotic agent;    -   the predetermined concentration is above the intermediate CLSI        MIC cutoff (for urine) for the antibiotic agent;    -   the predetermined concentration is above the resistant CLSI MIC        cutoff (for urine) for the antibiotic agent;    -   the predetermined concentration is at least 2-fold or greater        than the resistant CLSI MIC cutoff (for urine) for the        antibiotic agent;    -   the predetermined concentration is at least 4-fold or greater        than the resistant CLSI MIC cutoff (for urine) for the        antibiotic agent;    -   the predetermined concentration is between the intermediate CLSI        MIC cutoff and the resistant CLSI MIC cutoff (for urine) for the        antibiotic agent;    -   the predetermined concentration is below the sensitive CLSI MIC        cutoff (for urine) for the antibiotic agent;    -   the sensitive CLSI MIC cutoff (for urine) is at least 2-fold or        greater than the predetermined concentration for the antibiotic        agent;    -   the antibiotic agent comprises Gentamicin and the predetermined        concentration is between about 2 μg/mL and 16 μg/mL;    -   the predetermined concentration is between about 2 μg/mL and 4        μg/mL;    -   the predetermined concentration is about 2 μg/mL;    -   the predetermined concentration is about 4 μg/mL;    -   the sensitive CLSI MIC cutoff (for urine) for Gentamicin is        equal to or greater than the predetermined concentration;    -   the antibiotic agent comprises Ciprofloxacin and the        predetermined concentration is between about 1 μg/mL and 8        μg/mL;    -   The method of claim 178, wherein the predetermined concentration        is between about 1 μg/mL and 4 μg/mL;    -   the predetermined concentration is about 4 μg/mL;    -   the predetermined concentration is substantially equal to the        resistant CLSI MIC cutoff (for urine) for Ciprofloxacin;    -   the antibiotic agent comprises Cefazolin and the predetermined        concentration is between about 2 μg/mL and about 256 μg/mL;    -   the predetermined concentration is between about 16 μg/mL and        about 128 μg/mL;    -   the predetermined concentration is about 64 μg/mL;    -   the predetermined concentration is substantially equal to 2        times the resistant CLSI MIC cutoff (for urine) for Cefazolin;    -   the antibiotic agent comprises Ceftriaxone and the predetermined        concentration is between about 1 μg/mL and about 128 μg/mL;    -   the predetermined concentration is between about 16 μg/mL and        about 64 μg/mL;    -   the predetermined concentration is about 32 μg/mL;    -   the predetermined concentration is substantially equal to 8        times the resistant CLSI MIC cutoff (for urine) for Ceftriaxone;    -   the antibiotic agent comprises Cefepime and the predetermined        concentration is between about 4 μg/mL and about 128 μg/mL;    -   the predetermined concentration is between about 16 μg/mL and        about 128 μg/mL;    -   the predetermined concentration is between about 32 μg/mL and        about 64 μg/mL;    -   the predetermined concentration is about 32 μg/mL;    -   the predetermined concentration is about 64 μg/mL;    -   the predetermined concentration is substantially equal to 2 or 4        times the resistant CLSI MIC cutoff (for urine) for Cefepime;    -   the antibiotic agent comprises Ampicillin and the predetermined        concentration is between about 8 μg/mL and about 2048 μg/mL;    -   the predetermined concentration is between about 128 μg/mL and        about 512 μg/mL;    -   the predetermined concentration is about 128 μg/mL;    -   the predetermined concentration is about 512 μg/mL;    -   the predetermined concentration is substantially equal to about        4 times the resistant CLSI MIC cutoff (for urine) for        Ampicillin;    -   the predetermined concentration is substantially equal to about        16 times the resistant CLSI MIC cutoff (for urine) for        Ampicillin;    -   the antibiotic agent comprises Trimethoprim-Sulfamethoxazole and        the predetermined concentration for Trimethoprim is between        about 2 μg/mL and about 16 μg/mL and the predetermined        concentration for Sulfamethoxazole is between about 38 μg/mL and        about 304 μg/mL;    -   the predetermined concentration for Trimethoprim is between        about 4 μg/mL and about 8 μg/mL and the predetermined        concentration for Sulfamethoxazole is between about 76 μg/mL and        about 152 μg/mL;    -   the predetermined concentration for Trimethoprim is about 4        μg/mL and the predetermined concentration for Sulfamethoxazole        is about 76 μg/mL;    -   the predetermined concentration for        Trimethoprim-Sulfamethoxazole is substantially equal to the        resistant CLSI MIC cutoff (for urine) for        Trimethoprim-Sulfamethoxazole;    -   the antibiotic agent comprises Nitrofurantoin and the        predetermined concentration is between about 4 μg/mL and about        512 μg/mL;    -   the predetermined concentration is between about 8 μg/mL and        about 32 μg/mL;    -   the predetermined concentration is about 16 μg/mL;    -   the sensitive CLSI MIC cutoff (for urine) for Nitrofurantoin is        at least 2-fold or greater than the predetermined concentration;    -   the antibiotic agent comprises Fosfomycin and the predetermined        concentration is between about 4 μg/mL and about 512 μg/mL;    -   the predetermined concentration is between about 8 μg/mL and        about 128 μg/mL;    -   the predetermined concentration is about 64 μg/mL;    -   the sensitive CLSI MIC cutoff (for urine) for Fosfomycin is at        about equal to the predetermined concentration;    -   the antibiotic agent comprises Amoxicillin-Clavulanate and the        predetermined concentration for Amoxicillin is between about 2        μg/mL and about 256 μg/mL and the predetermined concentration        for Clavulanate is between about 1 μg/mL and about 128 μg/mL;    -   the predetermined concentration for Amoxicillin is between about        8 μg/mL and about 128 μg/mL and the predetermined concentration        for Clavulanate is between about 4 μg/mL and about 64 μg/mL;    -   the predetermined concentration for Amoxicillin is about 64        μg/mL and the predetermined concentration for Clavulanate is        about 32 μg/mL;    -   wherein the predetermined concentration for Amoxicillin is about        32 μg/mL and the predetermined concentration for Clavulanate is        about 16 μg/mL;    -   the predetermined concentration for Amoxicillin is about 16        μg/mL and the predetermined concentration for Clavulanate is        about 8 μg/mL;    -   the predetermined concentration is equal to the intermediate        CLSI MIC cutoff (for urine) for Amoxicillin-Clavulanate;    -   the predetermined concentration is greater than the intermediate        CLSI MIC cutoff (for urine) for Amoxicillin-Clavulanate;    -   the predetermined concentration is equal to or greater than the        resistant CLSI MIC cutoff (for urine) for        Amoxicillin-Clavulanate;    -   the antibiotic agent comprises Amikacin and the predetermined        concentration is between about 2 μg/mL and about 64 μg/mL;    -   the predetermined concentration is between about 8 μg/mL and        about 64 μg/mL;    -   the predetermined concentration is about 32 μg/mL;    -   the predetermined concentration is about 16 μg/mL;    -   the predetermined concentration is about 8 μg/mL;    -   the predetermined concentration is less than the resistant CLSI        MIC cutoff (for urine) for Amikacin;    -   the predetermined concentration is equal to the intermediate        CLSI MIC cutoff (for urine) for Amikacin;    -   the predetermined concentration is less than or equal to the        sensitive CLSI MIC cutoff (for urine) for Amikacin;    -   the antibiotic agent comprises Ertapenem and the predetermined        concentration is between about 0.5 μg/mL and about 8 μg/mL;    -   the predetermined concentration is between about 1 μg/mL and        about 4 μg/mL;    -   the predetermined concentration is about 4 μg/mL;    -   the predetermined concentration is about 2 μg/mL;    -   the predetermined concentration is greater than or equal to the        resistant CLSI MIC cutoff (for urine) for Ertapenem;    -   the antibiotic agent comprises Meropenem and the predetermined        concentration is between about 1 μg/mL and about 8 μg/mL;    -   the predetermined concentration is between about 1 μg/mL and        about 4 μg/mL;    -   the predetermined concentration is about 4 μg/mL;    -   the predetermined concentration is about 2 μg/mL;    -   the predetermined concentration is equal to the resistant CLSI        MIC cutoff (for urine) for Meropenem;    -   the predetermined concentration is equal to the intermediate        CLSI MIC cutoff (for urine) for Meropenem;    -   determining a baseline quantity of the nucleic acid molecule in        the control portion before the control incubation period is        complete and comparing the baseline quantity of the nucleic acid        molecule to the quantity of the nucleic acid molecule in the        control portion at the conclusion of the incubation period to        determine if the quantity of the nucleic acid in the control        portion increased by a measurement threshold amount during the        incubation period;    -   the microorganism comprises a Gram-negative bacterium;    -   the microorganism comprises a Gram-positive bacterium;    -   the microorganism is an unknown bacterium when steps a) to f) of        claim 139 are conducted; and    -   lysing the first test portion prior to determining the quantity        of the nucleic acid in the first test portion and lysing the        second test portion prior to determining the quantity of the        nucleic acid in the second test portion.    -   further comprising the steps of:        -   h) subjecting the first test portion and the second test            portion to mechanical lysis to cause disruption of a            cellular membrane in the microorganism in each;        -   i) contacting the first test portion and the second test            portion with an alkaline material to produce a first lysate            composition comprising the nucleic acid in the first test            portion and a second lysate composition comprising the            nucleic acid in the second test portion; and        -   j) recovering the first test portion lysate composition from            the first test portion and the second test portion lysate            composition from the second test portion.    -   Step i) comprises contacting the microorganisms in the first and        second test portions with an alkaline liquid;    -   Step i) comprises contacting the microorganisms in the first and        second test portions with an alkaline solution;    -   the alkaline solution is a sodium hydroxide solution;    -   the alkaline solution has a concentration of 10M or less;    -   the alkaline solution has a concentration in the range of from        1M to 5M;    -   the alkaline solution has a concentration in the range of from        1.5M to 3M;    -   the alkaline solution has a concentration of 2M;    -   the alkaline solution has a concentration of 3M;    -   lysing the first and second test portions comprises transferring        an aliquot of an inoculate from each of the first and second        test portion to a first and second lysing container;    -   incubating the first and second test portions is done within a        first and second test incubation chamber on a centrifugal disc,        and lysing the first and second test portions is conducted        within a first and second lysing chamber on the same centrifugal        disc;    -   the first lysing chambers is fluidly connected to the first test        incubation chamber and the second lysing chambers is fluidly        connected to the second test incubation chamber;    -   the first lysing chamber comprises the first test incubation        chamber and the second lysing chamber comprises the second test        chamber;    -   Steps h) and i) are conducted for a period of 10 minutes or        less;    -   Steps h) and i) are conducted for a period of from 30 seconds to        10 minutes;    -   Steps h) and i) are conducted for a period of from 1 minute to 8        minutes;    -   Steps h) and i) are conducted for a period of from 2 minutes±30        seconds;    -   Steps h) and i) are conducted for a period of from 3 minutes±30        seconds;    -   Steps h) and i) are conducted for a period of from 4 minutes±30        seconds;    -   Steps h) and i) are conducted for a period of from 5 minutes±30        seconds;    -   Steps h) and i) are conducted for a period of from 6 minutes±30        seconds;    -   Steps h) and i) are conducted for a period of from 7 minutes±30        seconds;    -   Steps h) and i) are carried out concurrently;    -   the mechanical lysis comprises a combination of centrifugation        and puck lysing;    -   the mechanical lysis comprises a combination of centrifugation        and magnetic puck lysing;    -   the combination of centrifugation and puck lysing is carried out        in a common lysis chamber;    -   Steps h) and i) are carried out concurrently;    -   Steps h) and i) are carried out sequentially;    -   Step i) is carried out after commencement of disruption of the        cellular membrane in Step h);    -   the microorganism is susceptible to the first antibiotic agent        if the quantity of the nucleic acid molecule in the control        portion is more than the quantity of the nucleic acid molecule        in the first test portion at the conclusion of the first        incubation period;    -   the microorganism is susceptible to the second antibiotic agent        if the quantity of the nucleic acid molecule in the control        portion is more than the quantity of the nucleic acid molecule        in the second test portion at the conclusion of the second        incubation period;    -   the microorganism is not susceptible to the first antibiotic        agent if the quantity of the nucleic acid molecule in the        control portion is nearly equal, equal, or less than the        quantity of the nucleic acid molecule in the first test portion        at the conclusion of the first incubation period;    -   the microorganism is not susceptible to the second antibiotic        agent if the quantity of the nucleic acid molecule in the        control portion is nearly equal, equal, or less than the        quantity of the nucleic acid molecule in the second test portion        at the conclusion of the second incubation period;    -   the microorganism is susceptible to the first antibiotic agent        when the quantity of the nucleic acid molecule in the first test        portion is about 40% or less of the quantity of the nucleic acid        molecule in the control portion at the conclusion of the first        incubation period;    -   the microorganism is susceptible to the second antibiotic agent        when the quantity of the nucleic acid molecule in the second        test portion is about 40% or less of the quantity of the nucleic        acid molecule in the control portion at the conclusion of the        second incubation period;    -   the microorganism is resistant to the first antibiotic agent        when the quantity of the nucleic acid molecule in the first test        portion is about 60% or more of the quantity of the nucleic acid        molecule in the control portion at the conclusion of the first        incubation period; and    -   the microorganism is resistant to the second antibiotic agent        when the quantity of the nucleic acid molecule in the second        test portion is about 60% or more of the quantity of the nucleic        acid molecule in the control portion at the conclusion of the        second incubation period.

In another of its aspects, the present invention relates to a method fordetermining the susceptibility of a microorganism in a sample to anantimicrobial agent, the method comprising: (a) inoculating a testportion of the sample in a medium containing a predeterminedconcentration of an antimicrobial agent; (b) inoculating a controlportion of the sample in a medium that does not contain theantimicrobial agent; (c) incubating the test portion and the controlportion for an incubation period that is less than 420 minutes; (d)determining a quantity of a nucleic acid molecule in the test portionand a quantity of the nucleic acid molecule in the control portion atthe conclusion of the incubation; and (e) determining a susceptibilityof the microorganism to the antimicrobial agent by comparing thequantity of the nucleic acid molecule in the test portion to thequantity of the nucleic acid molecule in the control portion.

Preferred embodiments of this method may include any one or acombination of any two or more of any of the following features:

-   -   the incubation period is equal to or less than 420 minutes;    -   the incubation period is equal to or less than 390 minutes;    -   the incubation period is equal to or less than 360 minutes;    -   the incubation period is equal to or less than 330 minutes;    -   the incubation period is equal to or less than 300 minutes;    -   the incubation period is equal to or less than 270 minutes;    -   the incubation period is equal to or less than 240 minutes;    -   the incubation period is equal to or less than 210 minutes;    -   the incubation period is equal to or less than 150 minutes;    -   the incubation period is equal to or less than 120 minutes;    -   the incubation period is equal to or less than 90 minutes;    -   the incubation period is equal to or less than 60 minutes;    -   the incubation period is equal to or less than 30 minutes;    -   the microorganism comprises prokaryotic cells;    -   the microorganism comprises bacteria;    -   the bacteria comprises Gram-negative bacteria;    -   the bacteria comprises Gram-positive bacteria;    -   the bacteria comprises an unknown bacterium when steps a) to f)        of claim 283 are conducted;    -   nucleic acid molecule comprises at least one of deoxyribonucleic        acid (DNA) and ribonucleic acid (RNA);    -   the nucleic acid molecule comprises RNA    -   the nucleic acid molecule comprises ribosomal RNA    -   the nucleic acid molecule comprises pre-ribosomal RNA,    -   the nucleic acid molecule comprises mature RNA;    -   the nucleic acid molecule comprises at least one of 16S rRNA and        23S rRNA;    -   the antimicrobial agent comprises at least one antibiotic agent;    -   the sample comprises an unknown cellular material;    -   the sample comprises mammalian cellular material;    -   the sample comprises human cellular material;    -   the sample comprises a bodily fluid;    -   the sample comprises an inoculant derived from a bodily fluid;    -   the bodily fluid is selected from the group consisting of blood,        urine, saliva, sweat, tears, mucus, breast milk, plasma, serum,        synovial fluid, pleural fluid, lymph fluid, amniotic fluid,        feces, cerebrospinal fluid, and any mixture of two or more of        these;    -   the bodily fluid is urine or an inoculant derived therefrom;    -   the bodily fluid is blood or an inoculant derived therefrom;    -   the antibiotic agent is a bactericidal antibiotic;    -   the antibiotic agent is a bacteriostatic antibiotic;    -   the antibiotic agent comprises at least one of Gentamicin,        Ciprofloxacin, Cefazolin, Ceftriaxone, Cefepime, Ampicillin,        Trimethoprim-Sulfamethoxazole, Nitrofurantoin, Fosfomycin,        Amoxicillin-Clavulanate, Amikacin, Ertapenem, Meropenem and        combinations thereof;    -   the predetermined concentration is above the sensitive CLSI MIC        cutoff (for urine) for the antibiotic agent;    -   the predetermined concentration is above the intermediate CLSI        MIC cutoff (for urine) for the antibiotic agent;    -   the predetermined concentration is above the resistant CLSI MIC        cutoff (for urine) for the antibiotic agent;    -   the predetermined concentration is at least 2-fold or greater        than the resistant CLSI MIC cutoff (for urine) for the        antibiotic agent;    -   the predetermined concentration is at least 4-fold or greater        than the resistant CLSI MIC cutoff (for urine) for the        antibiotic agent;    -   the predetermined concentration is between the intermediate CLSI        MIC cutoff and the resistant CLSI MIC cutoff (for urine) for the        antibiotic agent;    -   the predetermined concentration is below the sensitive CLSI MIC        cutoff (for urine) for the antibiotic agent;    -   the sensitive CLSI MIC cutoff (for urine) is at least 2-fold or        greater than the predetermined concentration for the antibiotic        agent;    -   the antibiotic agent comprises Gentamicin and the predetermined        concentration is between about 2 μg/mL and 16 μg/mL;    -   the predetermined concentration is between about 2 μg/mL and 4        μg/mL;    -   the predetermined concentration is about 2 μg/mL;    -   the predetermined concentration is about 4 μg/mL;    -   the sensitive CLSI MIC cutoff (for urine) for Gentamicin is        equal to or greater than the predetermined concentration of the        antibiotic agent;    -   the antibiotic agent comprises Ciprofloxacin and the        predetermined concentration is between about 1 μg/mL and 8        μg/mL;    -   The method of claim 333, wherein the predetermined concentration        is between about 1 μg/mL and 4 μg/mL;    -   the predetermined concentration is about 4 μg/mL;    -   the predetermined concentration is substantially equal to the        resistant CLSI MIC cutoff (for urine) for Ciprofloxacin;    -   the antibiotic agent comprises Cefazolin and the predetermined        concentration is between about 2 μg/mL and about 256 μg/mL;    -   the predetermined concentration is between about 16 μg/mL and        about 128 μg/mL;    -   the predetermined concentration is about 64 μg/mL;    -   the predetermined concentration is substantially equal to 2        times the resistant CLSI MIC cutoff (for urine) for Cefazolin;    -   the antibiotic agent comprises Ceftriaxone and the predetermined        concentration is between about 1 μg/mL and about 128 μg/mL;    -   the predetermined concentration is between about 16 μg/mL and        about 64 μg/mL;    -   the predetermined concentration is about 32 μg/mL;    -   the predetermined concentration is substantially equal to 8        times the resistant CLSI MIC cutoff (for urine) for Ceftriaxone;    -   the antibiotic agent comprises Cefepime and the predetermined        concentration is between about 4 μg/mL and about 128 μg/mL;    -   the predetermined concentration is between about 16 μg/mL and        about 128 μg/mL;    -   the predetermined concentration is between about 32 μg/mL and        about 64 μg/mL;    -   the predetermined concentration is about 32 μg/mL;    -   the predetermined concentration is about 64 μg/mL;    -   the predetermined concentration is substantially equal to 2 or 4        times the resistant CLSI MIC cutoff (for urine) for Cefepime;    -   the antibiotic agent comprises Ampicillin and the predetermined        concentration is between about 8 μg/mL and about 2048 μg/mL;    -   the predetermined concentration is between about 128 μg/mL and        about 512 μg/mL;    -   the predetermined concentration is about 128 μg/mL;    -   the predetermined concentration is about 512 μg/mL;    -   the predetermined concentration is substantially equal to about        4 times the resistant CLSI MIC cutoff (for urine) for        Ampicillin;    -   the predetermined concentration is substantially equal to about        16 times the resistant CLSI MIC cutoff (for urine) for        Ampicillin;    -   the antibiotic agent comprises Trimethoprim-Sulfamethoxazole and        the predetermined concentration for Trimethoprim is between        about 2 μg/mL and about 16 μg/mL and the predetermined        concentration for Sulfamethoxazole is between about 38 μg/mL and        about 304 μg/mL;    -   the predetermined concentration for Trimethoprim is between        about 4 μg/mL and about 8 μg/mL and the predetermined        concentration for Sulfamethoxazole is between about 76 μg/mL and        about 152 μg/mL;    -   the predetermined concentration for Trimethoprim is about 4        μg/mL and the predetermined concentration for Sulfamethoxazole        is about 76 μg/mL;    -   the predetermined concentration for        Trimethoprim-Sulfamethoxazole is substantially equal to the        resistant CLSI MIC cutoff (for urine) for        Trimethoprim-Sulfamethoxazole;    -   the antibiotic agent comprises Nitrofurantoin and the        predetermined concentration is between about 4 μg/mL and about        512 μg/mL;    -   the predetermined concentration is between about 8 μg/mL and        about 32 μg/mL;    -   the predetermined concentration is about 16 μg/mL;    -   the sensitive CLSI MIC cutoff (for urine) for Nitrofurantoin is        at least 2-fold or greater than the predetermined concentration;    -   the antibiotic agent comprises Fosfomycin and the predetermined        concentration is between about 4 μg/mL and about 512 μg/mL;    -   the predetermined concentration is between about 8 μg/mL and        about 128 μg/mL;    -   the predetermined concentration is about 64 μg/mL;    -   the sensitive CLSI MIC cutoff (for urine) for Fosfomycin is at        about equal to the predetermined concentration;    -   the antibiotic agent comprises Amoxicillin-Clavulanate and the        predetermined concentration for Amoxicillin is between about 2        μg/mL and about 256 μg/mL and the predetermined concentration        for Clavulanate is between about 1 μg/mL and about 128 μg/mL;    -   the predetermined concentration for Amoxicillin is between about        8 μg/mL and about 128 μg/mL and the predetermined concentration        for Clavulanate is between about 4 μg/mL and about 64 μg/mL;    -   the predetermined concentration for Amoxicillin is about 64        μg/mL and the predetermined concentration for Clavulanate is        about 32 μg/mL;    -   the predetermined concentration for Amoxicillin is about 32        μg/mL and the predetermined concentration for Clavulanate is        about 16 μg/mL;    -   the predetermined concentration for Amoxicillin is about 16        μg/mL and the predetermined concentration for Clavulanate is        about 8 μg/mL;    -   the predetermined concentration is equal to the intermediate        CLSI MIC cutoff (for urine) for Amoxicillin-Clavulanate;    -   the predetermined concentration is greater than the intermediate        CLSI MIC cutoff (for urine) for Amoxicillin-Clavulanate;    -   the predetermined concentration is equal to or greater than the        resistant CLSI MIC cutoff (for urine) for        Amoxicillin-Clavulanate;    -   the antibiotic agent comprises Amikacin and the predetermined        concentration is between about 2 μg/mL and about 64 μg/mL;    -   the predetermined concentration is between about 8 μg/mL and        about 64 μg/mL;    -   the predetermined concentration is about 32 μg/mL;    -   the predetermined concentration is about 16 μg/mL;    -   the predetermined concentration is about 8 μg/mL;    -   the predetermined concentration is less than the resistant CLSI        MIC cutoff (for urine) for Amikacin;    -   the predetermined concentration is equal to the intermediate        CLSI MIC cutoff (for urine) for Amikacin;    -   the predetermined concentration is less than or equal to the        sensitive CLSI MIC cutoff (for urine) for Amikacin;    -   the antibiotic agent comprises Ertapenem and the predetermined        concentration is between about 0.5 μg/mL and about 8 μg/mL;    -   the predetermined concentration is between about 1 μg/mL and        about 4 μg/mL;    -   the predetermined concentration is about 4 μg/mL;    -   the predetermined concentration is about 2 μg/mL;    -   the predetermined concentration is greater than or equal to the        resistant CLSI MIC cutoff (for urine) for Ertapenem;    -   the antibiotic agent comprises Meropenem and the predetermined        concentration is between about 1 μg/mL and about 8 μg/mL;    -   the predetermined concentration is between about 1 μg/mL and        about 4 μg/mL;    -   the predetermined concentration is about 4 μg/mL;    -   the predetermined concentration is about 2 μg/mL;    -   the predetermined concentration is equal to the resistant CLSI        MIC cutoff (for urine) for Meropenem;    -   the predetermined concentration is equal to the intermediate        CLSI MIC cutoff (for urine) for Meropenem;    -   determining a baseline quantity of the nucleic acid molecule in        the control sample before the incubation period is complete and        comparing the baseline quantity of the nucleic acid molecule to        the quantity of the nucleic acid molecule in the control portion        at the conclusion of the incubation period to determine if        quantity of the microorganism in the control portion increased        by a measurement threshold amount during the incubation period;    -   lysing the sample prior to determining a quantity of the nucleic        acid molecule in the test portion;    -   further comprising the steps of        -   f) subjecting the test portion to mechanical lysis to cause            disruption of a cellular membrane in the microorganism;        -   g) contacting the test portion with an alkaline material to            produce a lysate composition comprising the nucleic acid            molecule; and        -   h) recovering the lysate composition from the test portion.    -   Step g) comprises contacting the microorganism in the test        portion with an alkaline liquid;    -   Step g) comprises contacting the microorganism in the test        portion with an alkaline solution;    -   the alkaline solution is a sodium hydroxide solution;    -   the alkaline solution has a concentration of 10M or less;    -   the alkaline solution has a concentration in the range of from        1M to 5M;    -   the alkaline solution has a concentration in the range of from        1.5M to 3M;    -   the alkaline solution has a concentration of 2M;    -   the alkaline solution has a concentration of 3M;    -   lysing the test portion comprises transferring an aliquot of an        inoculate to a lysing container;    -   incubating the test portion is done within a test incubation        chamber on a centrifugal disc, and lysing the test portion is        conducted within a lysing chamber on the same centrifugal disc;    -   the lysing chamber is fluidically connected to the test        incubation chamber;    -   the lysing chamber comprises the test incubation chamber;    -   Steps f) and g) are conducted for a period of 10 minutes or        less;    -   Steps f) and g) are conducted for a period of from 30 seconds to        10 minutes;    -   Steps f) and g) are conducted for a period of from 1 minute to 8        minutes;    -   Steps f) and g) are conducted for a period of from 2 minutes±30        seconds;    -   Steps f) and g) are conducted for a period of from 3 minutes±30        seconds;    -   Steps f) and g) are conducted for a period of from 4 minutes±30        seconds;    -   Steps f) and g) are conducted for a period of from 5 minutes±30        seconds;    -   Steps f) and g) are conducted for a period of from 6 minutes±30        seconds;    -   Steps f) and g) are conducted for a period of from 7 minutes±30        seconds;    -   Steps f) and g) are carried out concurrently;    -   the mechanical lysis comprises a combination of centrifugation        and puck lysing;    -   the mechanical lysis comprises a combination of centrifugation        and magnetic puck lysing;    -   the combination of centrifugation and puck lysing is carried out        in a common lysis chamber;    -   Steps f) and g) are carried out concurrently;    -   Steps f) and g) are carried out sequentially;    -   Step g) is carried out after commencement of disruption of the        cellular membrane in Step f);    -   the microorganism is susceptible to the antibiotic agent if the        quantity of the nucleic acid molecule in the control portion is        more than the quantity of the nucleic acid molecule in the test        portion at the conclusion of the incubation period;    -   the microorganism is not susceptible to the antibiotic agent if        the quantity of the nucleic acid molecule in the control portion        is nearly equal, equal, or less than the quantity of the nucleic        acid molecule in the test portion at the conclusion of the        incubation period;    -   the microorganism is susceptible to the antibiotic agent when        the quantity of the nucleic acid molecule in the test portion is        about 40% or less of the quantity of the nucleic acid molecule        in the control portion at the conclusion of the incubation        period; and    -   the microorganism is resistant to the antibiotic agent when the        quantity of the nucleic acid molecule in the test portion is        about 60% or more of the quantity of the nucleic acid molecule        in the control portion at the conclusion of the incubation        period.

As used herein, certain terms may have the following defined meanings.

As used in the specification and claims, the singular form “a,” “an” and“the” include singular and plural references unless the context clearlydictates otherwise. For example, the term “a cell” includes a singlecell as well as a plurality of cells, including mixtures thereof.

As used in the specification and claims, the term “RiboResponse™” refersto the use of a nucleic acid molecule (such as a ribosomal ribonucleicacid (“rRNA”) molecule) from a microorganism for determining theresponse of a cell, such as a microorganism, to an agent, such as anantimicrobial agent. That is, a molecular quantification techniqueutilizing nucleic acid molecules. For instance, a RiboResponse™ methodfor determining the susceptibility of a microorganism to anantimicrobial agent may be based on comparing the quantity of the rRNAmolecules from a microorganism that has not been exposed to anantimicrobial agent to the quantity of the rRNA molecule from amicroorganism that has been exposed to an antimicrobial agent.

As used in the specification and claims, as explained further below, theterm “predetermined concentration” refers to an amount of anantimicrobial agent that is utilized in a test/assay to modify thetest/assay to help achieve one or more objectives, such as reducingand/or minimizing an incubation period length, providing apredetermined, targeted incubation period length or reducing and/orminimizing the amount of the antimicrobial agent required to perform thetest/assay in an acceptable manner.

For example, as is explained in more detail herein, at least one aspectof the teachings described herein is directed to conducting an assayusing a predetermined concentration of an antimicrobial agent that hasbeen selected to help achieve a predetermined assay objective. What thepredetermined concentration amount is can differ based on the differentobjectives to be achieved as described herein, but is generallyunderstood to be a concentration that is selected prior to initiating anassay to assist in performing the assay in an desired, targeted mannerand to help dictate at least one aspect of the assay incubation process(such as the incubation time and/or antimicrobial usage). Thepredetermined concentration that is utilized in a given embodiment ofthe methods described herein will be based on the particularantimicrobial agent used and the particular assay-related parameter thatis intended to be controlled/modified and may vary between embodimentsand for different particular antimicrobial agents.

For example, some of the embodiments described herein relate toconducting an AST assay using a predetermined concentration of anantibiotic agent that has been preselected to influence at least oneparameter of the incubation phase of the assay. In such embodiments, theconcentration of the antibiotic agent (or other antimicrobial agent) maybe selected to help alter the incubation time required to complete theassay.

Optionally, the objective of the user/operator may be to minimize theincubation time required for a given test/assay, so as to help obtainthe assay results in the shortest practical time period. Alternatively,instead of minimizing the incubation time for a given assay using agiven antibiotic, the objective of the user/operator may be to adjustthe incubation period to meet a pre-determined, target incubation time,such as between about 90-120 minutes. In some embodiments, this mayresult in a targeted incubation period that meets the desiredpre-determined target time limit but is actually longer than the minimumincubation time that could be achieved for that antibiotic using adifferent predetermined concentration. Accordingly, the predeterminedconcentration of an antibiotic agent that is selected by a user toprovide an incubation period of about 90-120 minutes may be differentthan the predetermined concentration that would be selected by the userto provide the minimum incubation time for the same antibiotic agent.

Different predetermined concentrations may be utilized to target thesame incubation period lengths when using different antimicrobialagents, as described herein. That is, a concentration of anantimicrobial agent that may differ from conventionally utilizedconcentrations for a given antimicrobial agent, and which ispre-selected to provide an incubation period, for the givenantimicrobial agent, that has a desired, or target, duration (i.e., 60minutes, 90 minutes, 120 minutes, etc.). Such concentrations can bereferred to as rate-targeted concentrations. Some examples ofpredetermined concentrations suitable for targeting a predeterminedincubation period length can include the concentrations described as“supratherapeutic” amounts as described in U.S. provisional patentapplication Ser. No. 62/547,361, filed Aug. 18, 2017 and EntitledMethods For Antimicrobial Susceptibility Testing, as well as theconcentrations described herein.

In other embodiments, the objective of modifying the test/assay may beto reduce the amount of the antimicrobial agent used/consumed during theprocess while still obtaining acceptably accurate test results, withoutemphasis on a specific or minimized incubation period length. In suchexamples, the predetermined concentration appropriate to achieve theobjective may differ from the predetermined concentrations that would beused if the objective was to minimize the incubation period or to targeta specific incubation period length.

As explained above, when implementing the methods described herein auser may decided on a particular objective to be achieved (incubationlength reduction, incubation length targeting or antimicrobial usagereduction) and based on the teachings herein may then select apredetermined concentration of a particular antimicrobial agent for usein the test/assay so as to help achieve the selected objective.

The terms “cell culture medium” and “cell culture media” used hereinrefer to a medium/media where a microorganism is capable of rapidgrowth. A cell culture medium may or may not contain at least oneantimicrobial agent. In some embodiments, a cell culture medium maycontain no antimicrobial agents. In some embodiments, a cell culturemedium may contain one antimicrobial agent. In some embodiments, a cellculture medium may contain more than one antimicrobial agents.

The terms “specimen” or “sample” used herein refers to a material whichis isolated from its natural environment, including but not limited tobiological materials (see definition of “clinical specimen” below), foodproducts, and fermented products.

The term “clinical specimen” used herein refers to samples of biologicalmaterial, including but not limited to urine, blood, serum, plasma,saliva, tears, gastric and/or digestive fluids, stool, mucus, sputum,sweat, earwax, oil, semen, vaginal fluid, glandular secretion, breastmilk, synovial fluid, pleural fluid, lymph fluid, amniotic fluid, feces,cerebrospinal fluid, wounds, burns, and tissue homogenates. The clinicalspecimen may be collected and stored by any means, including in asterile container.

A clinical specimen may be provided by or taken from any mammal,including but not limited to humans, dogs, cats, murines, simians, farmanimals, sport animals, and companion animals.

The term “incubation period” used herein refers to the period of timebetween when a sample is introduced into a test apparatus and allowed togrow, in the presence of a suitable media, and exposed to anantimicrobial agent (if a test sample) or not exposed to anantimicrobial agent (if a control sample) and when the growth period isstopped. The end of the incubation period may be the time at which agiven sample is observed for the purpose of determining the results ofthe growth, or when a further action is taken with the sample thatinhibits or stops the growth process. For example, in some of theexamples described herein a test portion of a sample can be incubatedduring an incubation period that begins when a sample portion isintroduced into a suitable incubation chamber and ends when the sampleportion is lysed to expose some target nucleic acid molecules forcounting/quantification.

The term “control portion” used herein refers to a portion of theclinical specimen which will not be exposed to an antimicrobial agent.In some embodiments, the control portion may include a plurality ofportions of the clinical specimen which will not be exposed to anantimicrobial agent.

The term “test portion” used herein refers to a portion of the clinicalspecimen which is to be exposed to at least one antimicrobial agent. Insome embodiments, the test portion may include a plurality of portionsof the clinical specimen which are to be exposed to at least oneantimicrobial agent. In some embodiments, the test portion may include1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more portions ofthe clinical specimen which are to be exposed to at least oneantimicrobial agent. In some embodiments, a single test portion isexposed to one antimicrobial agent.

The term “inoculate” used herein refers to the introduction of aclinical specimen, or a portion thereof, to a culture medium. Once aclinical specimen, or a portion thereof, has been introduced into aculture medium, it may also be referred to as “an inoculate”.

The term “bacteria” used herein refers to any species of bacteria,including but not limited to Gram-negative and Gram-positive bacteria,anaerobic bacteria, and parasites. In certain embodiments, the bacteriamay be Gram-negative bacteria, Gram-positive bacteria, or a mixturethereof. Examples of Gram-negative bacteria may include, but are notlimited to Escherichia coli, Salmonella, Shigella, Enterobaceriaceae,Pseudomonas, Moraxella, Helicobacter, Strenotrophomonas, Bdellovibrio,and Legionella. Examples of Gram-positive bacteria may include, but arenot limited to Enterococcus, Staphylococcus, Streptococcus, Actinomyces,Bacillus, Clostridium, Corynebacterium, Listeria, and Lactobacillus.

The term “bacterial density” used herein refers to the actualconcentration of bacteria in a specimen. Bacterial density is expressedherein in colony forming units per milliliter (CFU/ml) but can beexpressed by any other units, including but not limited to genomes permilliliter, ribosomal RNA per milliliter, or RNA molecules.

The term “bacterial density value” used herein refers to an estimate orapproximation of the bacterial concentration in a specimen. Thebacterial density value may refer to a species-specific concentration ofbacteria or may refer to the concentration of more than one species ofbacteria. Bacterial density value is expressed herein in colony formingunits per milliliter (CFU/mL) but can be expressed by any other units,including but not limited to genomes per milliliter, ribosomal RNA permilliliter, or RNA molecules.

The term “rRNA” used herein refers to the ribosomal ribonucleic acid ofbacteria present in a specimen.

The term “rRNA concentration” used herein refers to the number of rRNAmolecules per volume tested. rRNA concentration is expressed herein inpicomolar (pM) units but can be expressed by any another units.

The term “rRNA signal” used herein refers to the rRNA analyteconcentration determined by the quantification of rRNA concentration ina specimen. An rRNA signal can be quantified by any known or unknownplatform or method. Known platforms include but are not limited toelectrochemical sensor platforms, optical platforms (e.g. ELISA,magnetic beads, capture probe arrays), and qRT-PCR.

The term “positive control” used herein refers to a known concentrationof a target molecule that is included in an assay to produce a known andexpected effect. Examples of target molecules that can be used aspositive controls would be known to the person skilled the art, andinclude synthetic oligonucleotides that have the same sequence as thetarget rRNA sequence.

The term “negative control” used herein refers to a known treatment thatis included in an assay that is not expected to have any effect.Examples of treatments that can be used as negative controls would beknown to the person skilled the art, and include specimens that do notcontain rRNA, including RNase-treated samples.

The term “background” used herein refers to the result obtained fromsamples lacking rRNA, bacteria, or other microbes.

The term “infection threshold” used herein refers to the minimumbacterial density in a clinical specimen that indicates the presence ofinfection. A clinical specimen with a bacterial density above the“infection threshold” therefore may suggest the presence of infection.Bacterial densities below the cutoff may be considered negative forinfection, possibly indicating such factors as contamination of thespecimen during collection or outgrowth of contaminants during storageor transport. The infection threshold and how it is determined maydiffer for the type of specimen being analyzed, for the species ofbacteria being analyzed, and/or for the infection being tested for. Forexample, when assessing for the presence of a urinary tract infection, afalse negative rate of ≤5% may often be sufficient for tests forbacteriuria, which may be achieved by setting the infection threshold to2 standard deviations above background.

The term “target inoculation concentration” used herein refers to theconcentration of bacteria in a clinical specimen, or a range ofconcentrations of bacteria in a clinical specimen, that, when inoculatedinto growth medium, may provide accurate results on an AST. For example,for a direct from specimen phenotypic AST of a urine specimen, aninoculation concentration of ideally 5×10⁵ CFU/mL and of no greater than5×10⁶ CFU/mL may provide an accurate AST result, whereas inoculationconcentrations more than 5×10⁶ CFU/mL may reduce the accuracy. Thetarget inoculation concentration may be used to determine what dilutionfactor, if any, is required to dilute a clinical specimen such that thebacterial density of the specimen may be optimized for an AST.

As explained herein, a predetermined concentration may be different fordifferent antimicrobial agents. For some antimicrobial agents, a“predetermined concentration” may be above the minimum concentration ofthe antimicrobial agent that would be used therapeutically to treat asubject with an infection of a microorganism susceptible to theantimicrobial agent. For some antimicrobial agents, a “predeterminedconcentration” may be equal to the minimum concentration of theantimicrobial agent that would be used therapeutically to treat asubject with an infection of a microorganism susceptible to theantimicrobial agent. For some antimicrobial agents, a “predeterminedconcentration” may be below the minimum concentration of theantimicrobial agent that would be used therapeutically to treat asubject with an infection of a microorganism susceptible to theantimicrobial agent.

The predetermined concentration may, in some instances, be defined inrelation to the Clinical and Laboratory Standards Institute (CLSI)minimum inhibitory concentration (MIC) breakpoint for that antimicrobialagent. In some embodiments, a “predetermined concentration” of anantimicrobial agent is an amount (i.e., concentration) below thesusceptible CLSI MIC breakpoint for the antimicrobial agent. In someembodiments, a “predetermined concentration” of an antimicrobial agentis an amount (i.e., concentration) above the susceptible CLSI MICbreakpoint for the antimicrobial agent. In some embodiments, a“predetermined concentration” of an antimicrobial agent is an amountbetween the susceptible CLSI MIC breakpoint and the intermediate CLSIMIC breakpoint for the antimicrobial agent. In some embodiments, a“predetermined concentration” of an antimicrobial agent is an amountabove the intermediate CLSI MIC breakpoint for the antimicrobial agent.In some embodiments, a “predetermined concentration” of an antimicrobialagent is an amount between the intermediate CLSI MIC breakpoint and theresistant CLSI MIC breakpoint for the antimicrobial agent. In someembodiments, a “predetermined concentration” of an antimicrobial agentis an amount above the resistant CLSI MIC breakpoint for theantimicrobial agent.

The predetermined concentration(s) for a given antimicrobial agent maybe determined by empirical testing, and may be collected in a database,look-up table or the like which can then be used to determine aparticular predetermined concentration that should be used when tryingto achieve a particular objective, such as when manipulating theperformance of different antibiotic agents in a given testingcircumstance/environment so that the incubation period for eachantibiotic agent approaches the same target incubation period length. Inthe present case, the inventors have conducted tests on a variety ofdifferent antimicrobial agents and have identified a variety ofpotentially useful, predetermined concentrations. For example, Table 1below shows some examples of some predetermined concentrations that canbe used to achieve targeted incubation period lengths when the object ofthe test/assay is to provide an incubation period of about 90 minutesfor the antimicrobials listed:

TABLE 1 Predetermined concentration and MIC Criteria for selectantibiotics to provide an incubation period of about 90 mins. MICCriteria (μg/mL)¹ Predetermined S I R concentration(μg/mL) Ampicillin ≤816 ≥32 128 Cefazolin ≤2 4 ≥8 64 Ceftriaxone ≤1 2 ≥4 32 Cefepime ≤8 16≥32 64 ¹CLSI M100 (2018).

Table 2 below shows additional examples of some predeterminedconcentrations that can be used to define incubation periods for theantimicrobials listed, as determined by additional tests conducted bythe inventors in the present case:

TABLE 2 Predetermined concentration and MIC Criteria for selectantibiotics to provide an incubation period of about 90 mins. CLSI MICCutoffs for (Urine) AST Predetermined Determination and RiboResponseconcentration(μg/mL) Antibiotic Panels Working Working Antibiotic S SDDI R Panel 1 Panel 2 Amikacin ≤16 32 ≥64 64 32, 16, 8 Amox/Clav ≤8/4 16/8≥32/16 32/16 64/32, 32/16, 16/8 Ampicillin ≤8 16 ≥32 512 128 Cefazolin≤16 ≥32 64 64 Cefepime ≤2 4-8 ≥16 64 64, 32 Ceftriaxone ≤1 2 ≥4 32 32Ciprofloxacin ≤1 2 ≥4 4 4 Ertapenem ≤0.5 1 ≥2 4, 2 Fosfomycin ≤64 128≥256 64 64 Gentamicin ≤4 8 ≥16 4 4, 2 Imipenem ≤1 2 ≥4 4 Meropenem ≤1 2≥4 4, 2 Nitrofurantoin ≤32 64 ≥128 16 16 Pip/Tazo ≤16/4  32/4-64/4≥128/4  16/4  TMP/SMX  ≤2/38  ≥4/76  8/152 4/76 Amox/Clav =Amoxicillin/Clavulanate, Pip/Tazo = Piperacillin/Tazobactam, TMP/SMX =Trimethoprim/Sulfamethoxazole. CLSI AST Results: S = Susceptible, I =Intermediate, R = Resistant, SDD = Susceptible dose-dependent (treatmentwith this drug for this MIC range require higher than normal drugexposure).

The urine cutoffs for cefazolin (S<=16, R>=32) in Table 2 are for“uncomplicated” UTI cases as identified in the CLSI. The cutoffs forcefazolin from Table 1 are suitable for use with cefazolin cases notidentified as “uncomplicated”. Both cutoff ranges are listed in the CLSIM100 from 2018.

Accordingly, if a user was attempting to provide a commercially usableAST assay that could be performed with an incubation period of about 90minutes, and using one or more of the antibiotics listed above, thecommercial test apparatus could be pre-loaded with the correspondingpredetermined concentrations of the antibiotics described herein. If anAST is to be developed using antibiotics or targeting a particularmicroorganism that was not tested or described herein, a person skilledin the art could, based on the teachings here, replicate theexperimentation described and derive concentrations for the otherantibiotics or microorganisms that can provide an incubation period ofabout 90 minutes.

In these experiments, the first working panel of antibiotics wasdeveloped to help produce acceptably accurate AST answers/results withan incubation period of between about 60-90 minutes for E. coli. Whileit was found that this first panel was effective on other Gram-negativebacteria and time points, it was observed that using this panel withmicroorganisms that differed from E. coli with an incubation period ofabout 60-90 tended to reduce the accuracy of the AST results.

In subsequent experiments, a second working panel of agents was thendeveloped to help maintain the same level of AST determination accuracyacross a relatively wider variety of Gram negative pathogens andtargeting about 90-120 minutes incubation for both lab grown bacteriaand those from clinical urine specimens. It was discovered that theincreasing the incubation time was helped to extend the use of the panelto some types of relatively slower-growing bacteria and helped tofacilitate adequate growth in the antibiotic free control portions. Inthis example, ertapenem and meropenem replaced imipenem to help providea relatively wider coverage/detection of carbapenem resistantEnterobacteriaceae. This may also help bypass the long-term instabilityof imipenem in solution. Piperacillin/Tazobactam was dropped from thesecond working panel due to its relatively poor performance (accuracywas too low) when used in relation to non-E. coli bacteria at 90 and120-minute incubation periods, but could remain useful using longerincubation periods and/or in assays targeting E. coli.

During the development of present assay and the specific antibioticconcentrations listed in Table 2, the initial concentration consideredfor each given antibiotic was inclusive of the CLSI cutoffs. It wasunexpectedly discovered that the preferred rate-targeting concentrations(the working concentrations in the table) for at least some antibioticswould differ, sometimes substantially, from the CLSI cutoffs. Even moreunexpected was that some of the preferred rate-targeting concentrationswere below the respective susceptible cutoff and some were above theirrespective resistant cutoff. While the exact reason for thesedifferences may not be fully understood, it may be that it relates tothe specific mechanism of action of an antibiotic. For example, if theantibiotic acts relatively slowly on the bacterial cell, it may requirea relatively higher concentration to see an appropriate result fromsusceptible bacteria in the relatively rapid assay window or shortincubation periods. This may help reduce false resistance results.Conversely, if an antibiotic acts relatively quickly on the bacterialcell, it may require a relatively lower concentration it to obtain anaccurate result, and to help reduce the chances of an inappropriateresult.

It was also discovered that the rate-targeting dosages for almost all ofthe beta-lactam antibiotics tested in assays having incubation times ofbetween 60-120 minutes were concentrations above the respective CLSIresistant cutoffs. The fact that these antibiotics attack the bacteriaby inhibiting cell wall synthesis may be one factor that contributes tothe apparent advantage of providing relatively higher antibioticconcentrations to help achieve the desired incubation times. It was alsodiscovered that the folate synthesis inhibitor combination oftrimethoprim and sulfamethoxazole, which is bacteriostatic, also appearsto have a relatively higher rate-targeting concentration.

In contrast, some antibiotics, like Nitrofurantoin for example, werediscovered to have rate-targeting concentrations at or below thesusceptible CLSI cutoff when configured to achieve the desired60-120-minute incubation period. These concentrations were determined toprovide relatively accurate results in our selected incubation periods.It is noted that the traditional optical and microscopic techniques usedto generate and validate these CLSI cutoffs would involve incubatingwith concentrations that are too high for the targeted incubationperiod(s) for certain antibiotics, which could cause resistant bacteriato incorrectly appear susceptible. This may be because the traditionaltests incubate for a long enough time (several hours) with theantibiotic to help overcome any initial growth inhibition for theresistant bacteria and therefore generate reliable results. The oppositewould generally apply for antibiotics that have a working concentrationat or above the resistant CLSI cutoff. The traditional optical andmicroscopic techniques used to generate and validate these CLSI cutoffswould involves incubating with concentrations that are too low for thepresent methods and desired incubation period for certain antibiotics,and would cause susceptible bacteria to incorrectly appear resistant.The traditional tests incubate for enough time (several hours) with theantibiotic to overcome any initial growth for the susceptible bacteriaand generate reliable results. The unique combination of the ribosomalRNA assay and unconventional, and in some cases optimized, antibioticconcentrations help facilitate the providing an incubation period ofdesired length while maintaining an acceptable accuracy of the AST testresults.

In addition to the working panels summarized in Table 2, furtherexperiments were conducted to help identify some upper and lower boundson the rate-targeting concentration that can still help provide usefulresults, but may not be the rates that are most suitable for conductingthe assay within the 60-120-minute incubation period window. Table 3summaries the results of this testing:

TABLE 3 List of Highest and Lowest Concentrations Tested that providedacceptable AST assay results and the related concentration to provide a90-120-minute incubation period. Upper Lower Concen- Concen- 90-120tration tration Minute Antibiotic Limit Limit Incubation Amikacin 64 232, 16, 8 Amoxicillin/ 256/128 2/1 64/32, 32/16, 16/8 ClavulanateAmpicillin 2048 8 128  Cefazolin 256 2 64 Cefepime 128 4 64, 32Ceftriaxone 128 1 32 Ciprofloxacin 8 1  4 Ertapenem 8 0.5  4, 2Fosfomycin 512 4 64 Gentamicin 16 2 4 ,2 Meropenem 8 1 4, 2Nitrofurantoin 512 4 16 TMP/SMX 16/304 2/38 4/76 Concentrations arelisted in μg/ml; TMP/SMX = Trimethoprim/Sulfamethoxazole

If the objective of a given user is to minimize the incubation time whenusing one of the listed antibiotics, the values from column 1 may be thesuitable predetermined concentration. If the objective is to provide anincubation period of 90-120 minutes, the values from column 3 may be thesuitable predetermined concentration. If the objective is to consume theminimal amount of the antibiotic agent while still obtaining accurateresults, the values from column 2 may be the suitable predeterminedconcentration.

The methods disclosed herein comprise the use of one or more differentantimicrobial agents. Use of one or more antimicrobial agents maycomprise producing an inoculate comprising a microorganism in a cellculture media containing one or more antimicrobial agents. Use of one ormore antimicrobial agents may comprise obtaining an inoculate comprisinga microorganism in a cell culture media containing one or moreantimicrobial agents. Use of one or more antimicrobial agents maycomprise exposing a microorganism to one or more antimicrobial agents.

Testing was conducted on a variety of different antimicrobial agents tohelp identify one or more potentially useful concentrations for thedifferent agents. Based on this testing, which included the experimentsdescribed herein, a variety of different concentrations for differentantimicrobial agents, and for different testing objectives, werediscovered.

Based on these predetermined concentrations, one or more methods fordetermining the susceptibility of a microorganism in a sample to a givenantimicrobial agent can include the steps of dividing the sample into atleast one test portion and at least one control portion and incubatingthe test portion in the presence of the predetermined concentration andseparately incubating the control portion. At the end of the incubationperiod, both portions can be further processed if desired (such as vialysing) and the relative amounts of a target nucleic acid molecule (suchas DNA or RNA) in each of the portions can be determined. Themicroorganism can be considered to be susceptible to the particularantimicrobial agent if the concentration of the target nucleic acidmolecule in the test portion is below a susceptibility cutoff level orthreshold, can be considered to be resistant if the concentration of thetarget nucleic acid molecule in the test portion is above a resistantcutoff level or threshold, and may be considered indeterminate if theconcentration of the target nucleic acid molecule in the test portion isbetween the susceptibility and resistant thresholds.

For example, in some of the experiments discussed herein, thesusceptibility threshold was selected to be about 40% of theconcentration of the target nucleic acid molecule in the control portionand the resistant threshold was selected to be about 60% of theconcentration of the target nucleic acid molecule in the controlportion. That is, for example, a microorganism was considered to besusceptible to a given antimicrobial agent if the concentration of rRNAin the test portion was less than or equal to about 40% of theconcentration of rRNA in the antibiotic free control portion, resistantif the concentration of rRNA in the test portion was greater than orequal to about 60% of the concentration of rRNA in the antibiotic freecontrol, and the results were considered to be indeterminate if theconcentration of rRNA in the test portion was between about 40% andabout 60% of the concentration of rRNA in the antibiotic free control.

It was discovered that at least some of these threshold values may befurther refined for a given antibiotic agent and when being specificallyused in combination with an incubation period of between about 90-120minutes, while maintaining and/or enhancing accuracy (when compared tothe standard). This was found to be effective across a variety of gramnegative pathogens. Table 4 below summarize the experimental findingsrelated to the threshold values for certain, tested antibiotic agents.Such thresholds could be incorporated into an automated instrument'ssoftware program to help facilitate the automated interpretation of theresults by comparison to reference criteria.

TABLE 4 Antibiotic Specific-Cutoffs for RiboResponse AST Interpretation(% of Antibiotic Free) for (a) E. coli and (b) K. pneumoniae. (a) E.coli Amox/Clav Amp Cefaz Ceftriax Cipro Gent Nitro TMP/SMX Susceptible≤65 ≤55 ≤45 ≤65 ≤40 ≤65 ≤55 ≤69 Indeterminate 66-85 56-70 46-70 66-8041-70 66-80 56-60 70-75 Resistant ≥86 ≥71 ≥71 ≥81 ≥71 ≥81 ≥61 ≥76 (b) K.pneumoniae Amox/Clav Cefaz Cetriax Cipro Nitro TMP/SMX Susceptible ≤65≤55 ≤65 ≤35 ≤25 ≤65 Indeterminate 66-85 56-65 66-75 36-60 26-60 66-75Resistant ≥86 ≥66 ≥76 ≥61 ≥61 ≥76

To test the accuracy of the methods described herein, the assay wasconducted in parallel to the traditional (slow) method on blinded urinespecimens. Accuracy for this study was measured as how well theRiboResponse AST answer for a given specimen and antibiotic compared tothat of broth microdilution (through UCLA Clinical MicrobiologyLaboratory). By combining the ribosomal RNA assay with incrediblywell-optimized antibiotic concentrations, the rapid AST assay, utilizingRiboResponse, was able to generate results within hours of specimencollection, that normally take days, with 96% accuracy. It is believedthat a majority of the errors were caused by specimens containing amixture of susceptibility phenotypes, rather than a failure of the assaytechniques themselves. In these cases, the results measured the mixtureof the phenotypes present in the specimen and thus may still be used toinform treatment of the mixed infection in some cases.

As described herein, these threshold values were selected to yieldacceptable accuracy (when compared to the results obtained using brothmicrodilution) with the concentrations used to conduct the AST analysiswith incubation periods of only 90-120 minutes across a variety ofdifferent microorganisms (including different gram-negative pathogens).By way of example, the inventors have assessed the accuracy of thepresent invention (conducted with an incubation period of 90-120minutes) to determine susceptibility of microorganisms in a urinespecimen to a variety of antimicrobials, as compared to an AST conductedon the same samples using broth microdilution. These example results areset out in Table 5:

TABLE 5 Accuracy of Present Invention (i.e., RiboResponse-Based AST) forUrine Specimen Compared to the Conventional Testing Method Accuracy ofMinor Major Very Major Present Antibiotic Correct Error¹ Error² Error³Mixed⁴ Invention Amikacin 27 0 0 0 0 100.0% Amoxicillin/Clavulanate 23 10 0 0 95.8% Ampicillin 39 0 0 0 2 95.1% Cefazolin 41 0 0 0 1 97.6%Ceftriaxone 40 0 0 0 1 97.6% Ciprofloxacin 41 1 0 0 0 97.6% Gentamicin42 0 0 0 0 100.0% Nitrofurantoin 28 1 0 0 1 93.3%Trimethoprim/Sulfamethoxazole 38 0 1 0 3 90.5% Total 319 3 1 0 8 96.4%¹Minor error = Intermediate AST result from gold standard, ²Major error= False resistance from RiboResponse, ³Very major error = falsesusceptibility from RiboResponse, ⁴Mixed = specimens containing amixture of antibiotic susceptibility phenotypes (e.g., one specimencontaining two different E. coli strains, one susceptible toTrimethoprim/Sulfamethoxazole, one resistant).

Other ranges or thresholds may be used for different examples oradaptations of the methods described herein.

Inoculation

In some embodiments, the medium into which the test and/or controlportions of the clinical specimen are inoculated is in a container. Insome embodiments, the container is selected from the group of a tissueculture plate, vial, flask, microcentrifuge tube, and centrifugal disk.In some embodiments, the container is a well of a tissue culture plate.In some embodiments, the tissue culture plate contains a plurality ofwells (i.e., any number of wells). In some embodiments, the tissueculture plate contains 6, 12, 24, 48, 96, or more wells. In someembodiments, the container is a chamber of a centrifugal disc.

Optionally, multiple test chambers can be included in a singlecentrifugal disc, such that more than one test can be conducted using acommon apparatus, but preferably in fluid isolation from each other.

Optionally, more than one process step/phase can be conducted within acommon container or chamber within the container. For example, the stepsof inoculation, incubation and lysing may be performed in a singlechamber. This may help reduce the size and/or complexity of the testingapparatus and/or centrifugal disc. In such arrangements, the lysingagents/mechanisms, may be inactive during the incubation period orotherwise configured so as not to interrupt the incubation of a giventest portion until a desired processing time. For example, mechanicallysing agents may be held in a static position and/or chemical lysingagents may be encapsulated, segregated from the test portion duringincubation, introduced into the chamber at the conclusion of theincubation period or otherwise manipulated to only take effect at adesired time.

Incubation

In some embodiments, the one or more inoculates are shaken. In someembodiments, shaking an inoculate comprises placing the container withan inoculate in a shaking incubator. In some embodiments, shaking aninoculate comprises shaking the container with the inoculate at 400 ormore revolutions per minute (rpm). In some embodiments, shaking theinoculate occurs prior to determining the quantity of a nucleic acidmolecule in a plurality of inoculates.

In some embodiments, turbulence is generated by alternately acceleratingand decelerating the inoculate. In some embodiments, generatingturbulence comprises placing the container with the inoculate on arotating platform where the rotation alternately accelerates anddecelerates. In some embodiments, generating turbulence occurs prior todetermining the quantity of a nucleic acid molecule in a plurality ofinoculates. Methods for using a rotating platform for improving growthof a microorganism in a liquid culture have been disclosed inprovisional patent application Ser. No. 62/552,332, filed Aug. 30, 2017,the contents of which are hereby incorporated by reference herein in itsentirety.

In some embodiments, the inoculates are incubated at 23° C., 24° C., 25°C., 26° C., 27° C., 28° C., 29° C., 30° C., 31° C., 32° C., 33° C., 34°C., 35° C., 36° C., 37° C., 38° C., 39° C., 40° C., 41° C., or 42° C. Insome embodiments, the inoculates are incubated at 25° C. In someembodiments, the inoculates are incubated at 30° C. Preferably, theinoculates are incubated at about 37° C.

In some embodiments, the inoculates are incubated at suitabletemperatures for at least 30, 60, 90, 120, 150, 180, 210, 240, 270, 300,360, or 420 or more minutes. In some embodiments, the inoculates areincubated for at least 60 minutes. In some embodiments, the inoculatesare incubated for at least 90 minutes. In some embodiments, theinoculates are incubated for at least 120 minutes. In some embodiments,the inoculates are incubated for less than 420 minutes, less than 360minutes, less than 300 minutes, less than 270 minutes, less than 240minutes, less than 210 minutes, less than 180 minutes, less than 150minutes, less than 120 minutes, less than 90 minutes, less than 60minutes, or less than 30 minutes.

In some embodiments, the inoculates are incubated at 37° C. for at least30, 60, 90, 120, 150, 180, 210, 240, 270, 300, 360 or 420 or moreminutes. In some embodiments, the inoculates are incubated at 37° C. forat least 60 minutes. In some embodiments, the inoculates are incubatedat 37° C. for at least 90 minutes. In some embodiments, the inoculatesare incubated at 37° C. for at least 120 minutes. In some embodiments,the inoculates are incubated for less than 420 minutes, less than 360minutes, less than 300 minutes, less than 270 minutes, less than 240minutes, less than 210 minutes, less than 180 minutes, less than 150minutes, less than 120 minutes, less than 90 minutes, less than 60minutes, or less than 30 minutes. Preferably, the inoculates areincubated for less than 120 minutes,

Lysis

Methods for lysing the microorganism in an inoculate to produce a celllysate have been disclosed in PCT/US18/45211, which is incorporatedherein by reference herein in its entirety.

In some embodiments, the methods disclosed herein comprise steps forextracting a target chemical compound from a cellular material in asample, the steps comprising (a) subjecting the sample to mechanicallysis to cause disruption of a cellular membrane in the cellularmaterial; (b) contacting the sample with an alkaline material to producea lysate composition comprising the target chemical compound; and (c)recovering the lysate composition from the sample, wherein the targetchemical sample may be a nucleic acid. In some embodiments, the nucleicacid may be deoxyribonucleic acid (DNA). Examples of RNA involved inprotein synthesis include, but are not limited to, messenger RNA (mRNA),transfer RNA (tRNA), transfer-messenger RNA (tmRNA), single recognitionparticle RNA (SRP RNA), and ribosomal RNA (rRNA). In some embodiments,the nucleic acid may be ribonucleic acid (RNA). In certain preferredembodiments, the nucleic acid may be ribosomal RNA (rRNA), or morepreferably may pre-ribosomal rRNA, mature rRNA, or may be selected fromthe group consisting of 16S rRNA, 23S rRNA or any mixture thereof.

Provided in another embodiment is steps for extracting a target chemicalcompound from a cellular material in a sample, the steps comprising (a)subjecting the sample to mechanical lysis to cause disruption of acellular membrane in the cellular material; (b) contacting the samplewith an alkaline material to produce a lysate composition comprising thetarget chemical compound; and (c) recovering the lysate composition fromthe sample, wherein step (b) may comprise contacting the cellularmaterial in the sample with an alkaline solution. In some embodiments,the alkaline solution may be a sodium hydroxide solution. In certainpreferred embodiments, the alkaline solution may have a concentration ofabout 10M or less, preferably of about 1M to 5M, and more preferably ofabout 1.5M to 3M. In certain preferred embodiments, the alkalinesolution may have a concentration of about 2M. In other preferredembodiments, the alkaline solution may have a concentration of about 3M.

Provided in another embodiment is steps for extracting a target chemicalcompound from a cellular material in a sample, the method comprising (a)subjecting the sample to mechanical lysis to cause disruption of acellular membrane in the cellular material; (b) contacting the samplewith an alkaline material to produce a lysate composition comprising thetarget chemical compound; and (c) recovering the lysate composition fromthe sample, wherein the cellular material may be an unknown cellularmaterial.

Provided in another embodiment is steps for extracting a target chemicalcompound from a cellular material in a sample, the steps comprising (a)subjecting the sample to mechanical lysis to cause disruption of acellular membrane in the cellular material; (b) contacting the samplewith an alkaline material to produce a lysate composition comprising thetarget chemical compound; and (c) recovering the lysate composition fromthe sample, wherein the cellular material may be either a microorganism,prokaryotic cells, virally infected cells, fungus cells, or yeast cells.Examples of yeast cells may include but are not limited to Candidacells. Methods for detecting the presence of a fungal organisms within abiological sample, such as yeast have been disclosed in InternationalPatent Publication No. WO 2013166460 and WO 2015013324, both of whichare incorporated herein by reference herein in their entirety.

Provided in another embodiment is steps for extracting a target chemicalcompound from a cellular material in a sample, the steps comprising (a)subjecting the sample to mechanical lysis to cause disruption of acellular membrane in the cellular material; (b) contacting the samplewith an alkaline material to produce a lysate composition comprising thetarget chemical compound; and (c) recovering the lysate composition fromthe sample, wherein the cellular material may be bacteria.

Provided in another embodiment is steps for extracting a target chemicalcompound from a cellular material in a sample, the steps comprising (a)subjecting the sample to mechanical lysis to cause disruption of acellular membrane in the cellular material; (b) contacting the samplewith an alkaline material to produce a lysate composition comprising thetarget chemical compound; and (c) recovering the lysate composition fromthe sample, wherein the sample may comprise mammalian cellular material,preferably human cellular material, and more preferably a bodily fluidor an inoculant derived therefrom. In certain preferred embodiments, thebodily fluid may be selected from the group consisting of blood, urine,saliva, sweat, tears, mucus, breast milk, plasma, serum, synovial fluid,pleural fluid, lymph fluid, amniotic fluid, feces, cerebrospinal fluidand any mixture of two or more of these. Other examples of mammaliancellular material include but are not limited to samples from monkeys,cats, dogs, sheep, goats, cows, pigs, horses, or rabbits.

Provided in another embodiment is a method for extracting a targetchemical compound from a cellular material in a sample, the methodcomprising (a) subjecting the sample to mechanical lysis to causedisruption of a cellular membrane in the cellular material; (b)contacting the sample with an alkaline material to produce a lysatecomposition comprising the target chemical compound; and (c) recoveringthe lysate composition from the sample, wherein after disruption of thecellular membrane in the cellular material, the sample may be subjectedto biological lysis. In some embodiments, the biological lysis mayinclude contacting the sample with an enzyme. In certain preferredembodiments, the enzyme may be selected from the group consisting oflysozyme, lysostaphin and any mixture thereof.

Provided in another embodiment is a method for extracting a targetchemical compound from a cellular material in a sample, the methodcomprising (a) subjecting the sample to mechanical lysis to causedisruption of a cellular membrane in the cellular material; (b)contacting the sample with an alkaline material to produce a lysatecomposition comprising the target chemical compound; and (c) recoveringthe lysate composition from the sample, wherein after disruption of thecellular membrane in the cellular material, the sample may be subjectedto physical lysis. In some embodiments, the physical lysis may beselected from the group consisting of heating, osmotic shock, cavitationor any combination of two or more of these. Physical lysis methods suchas those mentioned above are common in the art. For example, lysis byheating may comprise placing the sample in a water bath, heat block, ortemperature controlled container, where the temperature of the waterbath, heat block, or temperature controlled container may be less thanor equal to about 100° C., preferably between about 40° C. and about100° C., or more preferably the sample may be heated at 45° C., 50° C.,55° C., 60° C., 65° C., 70° C., 75° C., 80° C., 85° C., 90° C., or 95°C. Cavitation may comprise nitrogen cavitation which may be performed by(a) placing cells from a sample in a pressure vessel; (b) dissolvingoxygen-free nitrogen in the cells under high pressure; and (c) releasingthe pressure in the vessel. Osmotic shock may be performed by changingthe concentration of a salt, substrate or solute around cells from asample, such that the cells rupture and/or release intracellularmaterials, such as nucleic acid molecules and proteins.

Provided in another embodiment is a method for extracting a targetchemical compound from a cellular material in a sample, the methodcomprising (a) subjecting the sample to mechanical lysis to causedisruption of a cellular membrane in the cellular material; (b)contacting the sample with an alkaline material to produce a lysatecomposition comprising the target chemical compound; and (c) recoveringthe lysate composition from the sample, wherein step (a) may beconducted for a period of about 10 minutes or less, preferably fromabout 30 seconds to about 10 minutes, more preferably from about 1minute to 8 minutes, and most preferably for a period of about 2minutes±30 seconds, about 3 minutes±30 seconds, about 4 minutes±30seconds, about 5 minutes±30 seconds, about 6 minutes±30 seconds, orabout 7 minutes±30 seconds.

Provided in another embodiment is a method for extracting a targetchemical compound from a cellular material in a sample, the methodcomprising (a) subjecting the sample to mechanical lysis to causedisruption of a cellular membrane in the cellular material; (b)contacting the sample with an alkaline material to produce a lysatecomposition comprising the target chemical compound; and (c) recoveringthe lysate composition from the sample, wherein the mechanical lysis maybe selected from the group consisting of French press, shaking,grinding, bead beating, centrifugation and any combination of two ormore of these. For example, lysis by French press may performed bypassing a sample through a narrow valve under high pressure. Lysis bygrinding may be performed by placing a sample in a grinder. Examples ofgrinders may include, but are not limited to, a ball mill, coffeegrinder, Geno/Grinder, and Retsch Mixer Mill. A ball mill for instance,may comprise a hollow cylindrical shell and one or more balls, where theballs may be made of chrome steel, stainless steel, ceramic, or rubber.Lysis by grinding may comprise, for example, the use of a mortar andpestle. Lysis by shaking may comprise, for example, mixing the samplewith some sort of bead or matrix, and placing the sample on a violenthigh-speed shaker.

In some embodiments, where the mechanical lysis is performed by beadbeating, said bead beating my comprise beating the sample with ceramicbeads, glass beads, zirconium beads, silica-zirconium beads, steel beadsor any combination of two or more of these. In certain preferredembodiments, bead beating may comprise the use of magnetic beads. By wayof non-limiting example, silica-zirconium beads may be preferable foruse in the disclose inventions as they are chemically inert and havebeen shown not to interfere with the assay techniques.

Provided in another embodiment is a method for extracting a targetchemical compound from a cellular material in a sample, the methodcomprising (a) subjecting the sample to mechanical lysis to causedisruption of a cellular membrane in the cellular material; (b)contacting the sample with an alkaline material to produce a lysatecomposition comprising the target chemical compound; and (c) recoveringthe lysate composition from the sample, wherein the mechanical lysis maycomprise using OmniLyse® or a functional equivalent thereof. Mechaniclysis with OmniLyse® or a functional equivalent thereof, for instance,may comprise the use of a small chamber containing, for example,zirconium beads, where the chamber is then connected to a syringe and amotor. By way of non-limiting example, OmniLyse® lysis may comprisedrawing a solution into the chamber with the syringe and turning on themotor to move the beads around at around 30,000 rpm with a smallpropeller, then ejecting the solution back into a tube using thesyringe.

Provided in another embodiment is a method for extracting a targetchemical compound from a cellular material in a sample, the methodcomprising (a) subjecting the sample to mechanical lysis to causedisruption of a cellular membrane in the cellular material; (b)contacting the sample with an alkaline material to produce a lysatecomposition comprising the target chemical compound; and (c) recoveringthe lysate composition from the sample, wherein the mechanical lysis maycomprise a combination of centrifugation and puck lysing. In someembodiments, the puck lysing may be magnetic puck lysing. In certainpreferred embodiments, the combination of centrifugation and disk lysingmay be carried out in a common lysis chamber, where preferablycentrifugation and puck lysing may be carried out on a centrifugal disk(CD). By way of non-limiting example, the centrifugal disk may compriseone or more microfluidic lysis chambers connected to one another by oneor more microfluidic channels, where at least one of the microfluidiclysis chambers has an inlet port which may be configured to receive afluid sample. Each lysis chamber of the CD may contain one or moremagnetic lysis pucks and a series of beads, wherein the lysis pucks andbeads are small enough to be able to move within the lysis chamber, butnot small enough to exit the lysis chamber through any of themicrofluidic channels. The CD may be configured to fit on a rotatingplatform connected to a motor, such that when the CD is placed on theplatform and the motor is turned on, the CD will rotate. The platform myfurther comprise a series of stationary magnets which may be configuredsuch that when the CD is rotating, the interaction between thestationary magnets and the magnetic lysis pucks causes the lysis pucksto move back and forth within each of the one or more lysis chambers.Lysis methods such as this are known in the art, including thosedisclosed in U.S. Pat. No. 8,303,911 which is incorporated by referenceherein in its entirety.

Provided in another embodiment is a method for extracting a targetchemical compound from a cellular material in a sample, the methodcomprising (a) subjecting the sample to mechanical lysis to causedisruption of a cellular membrane in the cellular material; (b)contacting the sample with an alkaline material to produce a lysatecomposition comprising the target chemical compound; and (c) recoveringthe lysate composition from the sample, wherein steps (a) and (b) may becarried out concurrently.

Provided in another embodiment is a method for extracting a targetchemical compound from a cellular material in a sample, the methodcomprising (a) subjecting the sample to mechanical lysis to causedisruption of a cellular membrane in the cellular material; (b)contacting the sample with an alkaline material to produce a lysatecomposition comprising the target chemical compound; and (c) recoveringthe lysate composition from the sample, wherein steps (a) and (b) may becarried out sequentially. In certain preferred embodiments, step (b) maybe carried out after commencement of disruption of the cellular membranein step (a). This sequential method may be preferred because alkalinelysing alone will not be able to disrupt the cellular membrane ofGram-positive cells and/or fungal cells. Thus, in order to get access tothe target compound within a Gram-positive and/or fungal cell, thecellular membrane is disrupted by the shear forces of mechanical lysing.

Provided in another embodiment is a method for extracting a targetchemical compound from a cellular material in a sample, the methodcomprising (a) subjecting the sample to mechanical lysis to causedisruption of a cellular membrane in the cellular material; (b)contacting the sample with an alkaline material to produce a lysatecomposition comprising the target chemical compound; and (c) recoveringthe lysate composition from the sample, wherein the method furthercomprises neutralizing the sample by contacting the sample with a buffersolution. When a sample is contacted with an alkaline solution, highconcentrations of hydroxide ions break apart the protein components of acell ribosome, unwind the secondary structure of rRNA, and break it intopieces. If this process is left unchecked, it will eventually break downthe entire rRNA into single bases. In order to arrest this process, aconcentrated buffer solution may be added to neutralize the pH of thelysate. In some embodiments, the buffer solution may be a phosphatebuffer solution. In certain preferred embodiments the buffer solutionmay have a pH of less than 7, preferably in the range of about 5 to 7.5,and more preferably in the range of 6 to 7.

Provided in another embodiment is a method for extracting a targetchemical compound from a cellular material in a sample, the methodcomprising (a) subjecting the sample to mechanical lysis to causedisruption of a cellular membrane in the cellular material; (b)contacting the sample with an alkaline material to produce a lysatecomposition comprising the target chemical compound; and (c) recoveringthe lysate composition from the sample, wherein the method furthercomprises contacting the sample with a nuclease inhibitor. In someembodiments, the sample may be contacted with a nuclease inhibitor priorto step (a). In certain preferred embodiment, the nuclease inhibitor maybe an RNAse inhibitor. For example, the RNAse inhibitor may be selectedfrom but is not limited to 2′-cytidine monophosphate free acid (2′-CMP),aluminon, adenosine 5′-pyrophosphate, 5′-diphosphoadenosine 3′-phosphate(ppA-3′-p), 5′-diphosphoadenosine 2′-phosphate (ppA-2′-p), Leucine,poly-L-aspartic acid, tyrosine-glutamic acid polymer, oligovinysulfonicacid, 5′-phospho-2′-deoxyuridine 3′-pyrophosphate P′→5′-ester withadenosine 3′-phosphate (pdUppAp).

Provided in another embodiment is a method for extracting a targetchemical compound from a cellular material in a sample, the methodcomprising (a) subjecting the sample to mechanical lysis to causedisruption of a cellular membrane in the cellular material; (b)contacting the sample with an alkaline material to produce a lysatecomposition comprising the target chemical compound; and (c) recoveringthe lysate composition from the sample, wherein the method furthercomprises detecting at least one nucleotide sequence in the cell lysate.In some embodiments, one or more nucleotide sequence may be detectedusing a sandwich assay, preferably where the sandwich assay is conductedon an electrochemical sensor platform. In certain preferred embodiments,one or more nucleotide sequences may be detected by contacting the celllysate with a capture probe. In other preferred embodiments, one or morenucleotide sequences may be detected by contacting the cell lysate witha magnetic bead, preferably where the magnetic bead comprises a captureprobe or a detector probe. In certain preferred embodiments, the captureprobe or detector probe may comprise one or more nucleic acids, examplesof which may include but are not limited to DNA, peptide nucleic acids(PNAs), locked nucleic acids (LNAs) or any combination thereof. By wayof non-limiting example, the capture probes and detector probes may eachcomprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20 or more nucleic acids. In further preferred embodiments, thedetector probe may comprise a detectable label. By way of non-limitingexample, the detectable label may be selected from a radionuclide, anenzymatic label, a chemiluminescent label, a hapten, and a fluorescentlabel. A fluorescent label for example, may be a fluorescent moleculeselected from a fluorophore, a cyanine dye, and a near infrared (NIR)dye, or more preferably the fluorescent molecule may be fluorescein orfluorescein isothiocyanate (FITC). A hapten label may for example beselected from DCC, biotin, nitropyrazole, thiazolesulfonamide,benzofurazan, and 2-hydroxyquinoxaline.

In another of its aspects, the present invention provides a method forproducing a lysate composition comprising RNA from a sample of mammalianorigin comprising a cellular material, the method comprising the stepsof: (a) rotating a microfluidic centrifugal disk comprising a lysischamber containing the sample; (b) subjecting the sample to mechanicallysis to cause disruption of a cellular membrane in the cellularmaterial; and (c) contacting the sample in the lysis chamber with analkaline solution to produce the lysate composition.

Provided in one embodiment is a method for producing a lysatecomposition comprising RNA from a sample of mammalian origin comprisinga cellular material, the method comprising the steps of: (a) rotating amicrofluidic centrifugal disk comprising a lysis chamber containing thesample; (b) subjecting the sample to mechanical lysis to causedisruption of a cellular membrane in the cellular material; and (c)contacting the sample in the lysis chamber with an alkaline solution toproduce the lysate composition, wherein the RNA may pre-ribosomal RNA,mature RNA, or may be selected from the group consisting of 16S rRNA,23S rRNA or any mixture thereof.

Provided in another embodiment is a method for producing a lysatecomposition comprising RNA from a sample of mammalian origin comprisinga cellular material, the method comprising the steps of: (a) rotating amicrofluidic centrifugal disk comprising a lysis chamber containing thesample; (b) subjecting the sample to mechanical lysis to causedisruption of a cellular membrane in the cellular material; and (c)contacting the sample in the lysis chamber with an alkaline solution toproduce the lysate composition, wherein the alkaline solution maycomprise a sodium hydroxide solution. In certain preferred embodiments,the alkaline solution may have a concentration of about 10M or less,preferably of about 1M to 5M, and more preferably of about 1.5M to 3M.In certain preferred embodiments, the alkaline solution may have aconcentration of about 2M. In other preferred embodiments, the alkalinesolution may have a concentration of about 3M.

Provided in another embodiment is a method for producing a lysatecomposition comprising RNA from a sample of mammalian origin comprisinga cellular material, the method comprising the steps of: (a) rotating amicrofluidic centrifugal disk comprising a lysis chamber containing thesample; (b) subjecting the sample to mechanical lysis to causedisruption of a cellular membrane in the cellular material; and (c)contacting the sample in the lysis chamber with an alkaline solution toproduce the lysate composition, wherein the sample may comprise humancellular material, preferably a bodily fluid or an inoculant derivedtherefrom. In certain preferred embodiments, the bodily fluid may beselected from the group consisting of blood, urine, saliva, sweat,tears, mucus, breast milk, plasma, serum, synovial fluid, pleural fluid,lymph fluid, amniotic fluid, feces, cerebrospinal fluid and any mixtureof two or more of these.

Provided in another embodiment is a method for producing a lysatecomposition comprising RNA from a sample of mammalian origin comprisinga cellular material, the method comprising the steps of: (a) rotating amicrofluidic centrifugal disk comprising a lysis chamber containing thesample; (b) subjecting the sample to mechanical lysis to causedisruption of a cellular membrane in the cellular material; and (c)contacting the sample in the lysis chamber with an alkaline solution toproduce the lysate composition, wherein steps (a) and (b) may beconducted for a period of about 10 minutes or less, preferably fromabout 30 seconds to about 10 minutes, more preferably from about 1minute to 8 minutes, and most preferably for a period of about 2minutes±30 seconds, about 3 minutes±30 seconds, about 4 minutes±30seconds, about 5 minutes±30 seconds, about 6 minutes±30 seconds, orabout 7 minutes±30 seconds.

Provided in another embodiment is a method for producing a lysatecomposition comprising RNA from a sample of mammalian origin comprisinga cellular material, the method comprising the steps of: (a) rotating amicrofluidic centrifugal disk comprising a lysis chamber containing thesample; (b) subjecting the sample to mechanical lysis to causedisruption of a cellular membrane in the cellular material; and (c)contacting the sample in the lysis chamber with an alkaline solution toproduce the lysate composition, wherein steps (a) and (b) may be carriedout concurrently.

Provided in another embodiment is a method for producing a lysatecomposition comprising RNA from a sample of mammalian origin comprisinga cellular material, the method comprising the steps of: (a) rotating amicrofluidic centrifugal disk comprising a lysis chamber containing thesample; (b) subjecting the sample to mechanical lysis to causedisruption of a cellular membrane in the cellular material; and (c)contacting the sample in the lysis chamber with an alkaline solution toproduce the lysate composition, wherein steps (b) and (c) may be carriedout concurrently.

Provided in another embodiment is a method for producing a lysatecomposition comprising RNA from a sample of mammalian origin comprisinga cellular material, the method comprising the steps of: (a) rotating amicrofluidic centrifugal disk comprising a lysis chamber containing thesample; (b) subjecting the sample to mechanical lysis to causedisruption of a cellular membrane in the cellular material; and (c)contacting the sample in the lysis chamber with an alkaline solution toproduce the lysate composition, wherein steps (b) and (c) may be carriedout sequentially. In certain preferred embodiments, step (c) may becarried out after commencement of disruption of the cellular membrane instep (b).

Provided in another embodiment is a method for producing a lysatecomposition comprising RNA from a sample of mammalian origin comprisinga cellular material, the method comprising the steps of: (a) rotating amicrofluidic centrifugal disk comprising a lysis chamber containing thesample; (b) subjecting the sample to mechanical lysis to causedisruption of a cellular membrane in the cellular material; and (c)contacting the sample in the lysis chamber with an alkaline solution toproduce the lysate composition, wherein the mechanical lysis maycomprise a combination of centrifugation and puck lysing. In someembodiments, the puck lysing may be magnetic puck lysing. In certainpreferred embodiments, the combination of centrifugation and puck lysingmay be carried out in a common lysis chamber, preferably centrifugationand puck lysing may be carried out on a centrifugal disk.

In yet another of its aspects, the present invention provides a methodfor extracting a nucleic acid from a cellular material in a samplecomprising a bodily fluid or an inoculant derived therefrom, the methodcomprising the steps of (a) subjecting the sample to a first lysingprocess comprising mechanical lysis to cause disruption of a cellularmembrane in the cellular material; (b) subjecting the sample to a secondlysing process comprising at least one of physical lysis, chemicallysis, biological lysis and any combination of two or more of these toproduce a lysate composition comprising the nucleic acid; and (c)recovering the lysate composition from the sample.

Provided in one embodiment is a method for extracting a nucleic acidfrom a cellular material in a sample comprising a bodily fluid or aninoculant derived therefrom, the method comprising the steps of (a)subjecting the sample to a first lysing process comprising mechanicallysis to cause disruption of a cellular membrane in the cellularmaterial; (b) subjecting the sample to a second lysing processcomprising at least one of physical lysis, chemical lysis, biologicallysis and any combination of two or more of these to produce a lysatecomposition comprising the nucleic acid; and (c) recovering the lysatecomposition from the sample, wherein the nucleic acid may bedeoxyribonucleic acid (DNA) or ribonucleic acid (RNA). In certainpreferred embodiments, the nucleic acid may be ribosomal RNA, or morepreferably may pre-ribosomal RNA, mature RNA, or may be selected fromthe group consisting of 16S rRNA, 23S rRNA or any mixture thereof.

Provided in another embodiment is a method for extracting a nucleic acidfrom a cellular material in a sample comprising a bodily fluid or aninoculant derived therefrom, the method comprising the steps of (a)subjecting the sample to a first lysing process comprising mechanicallysis to cause disruption of a cellular membrane in the cellularmaterial; (b) subjecting the sample to a second lysing processcomprising at least one of physical lysis, chemical lysis, biologicallysis and any combination of two or more of these to produce a lysatecomposition comprising the nucleic acid; and (c) recovering the lysatecomposition from the sample, wherein the chemical lysis may comprisecontacting the sample with an alkaline solution. In some embodiments,the alkaline solution may comprise a sodium hydroxide solution. Incertain preferred embodiments, the alkaline solution may have aconcentration of about 10M or less, preferably of about 1M to 5M, andmore preferably of about 1.5M to 3M. In certain preferred embodiments,the alkaline solution may have a concentration of about 2M. In otherpreferred embodiments, the alkaline solution may have a concentration ofabout 3M.

Provided in another embodiment is a method for extracting a nucleic acidfrom a cellular material in a sample comprising a bodily fluid or aninoculant derived therefrom, the method comprising the steps of (a)subjecting the sample to a first lysing process comprising mechanicallysis to cause disruption of a cellular membrane in the cellularmaterial; (b) subjecting the sample to a second lysing processcomprising at least one of physical lysis, chemical lysis, biologicallysis and any combination of two or more of these to produce a lysatecomposition comprising the nucleic acid; and (c) recovering the lysatecomposition from the sample, wherein the bodily fluid may comprise humancellular material, and more preferably may be selected from the groupconsisting of blood, urine, saliva, sweat, tears, mucus, breast milk,plasma, serum, synovial fluid, pleural fluid, lymph fluid, amnioticfluid, feces, cerebrospinal fluid and any mixture of two or more ofthese.

Provided in another embodiment is a method for extracting a nucleic acidfrom a cellular material in a sample comprising a bodily fluid or aninoculant derived therefrom, the method comprising the steps of (a)subjecting the sample to a first lysing process comprising mechanicallysis to cause disruption of a cellular membrane in the cellularmaterial; (b) subjecting the sample to a second lysing processcomprising at least one of physical lysis, chemical lysis, biologicallysis and any combination of two or more of these to produce a lysatecomposition comprising the nucleic acid; and (c) recovering the lysatecomposition from the sample, wherein step (a) may be conducted for aperiod of about 10 minutes or less, preferably from about 30 seconds toabout 10 minutes, more preferably from about 1 minute to 8 minutes, andmost preferably for a period of about 2 minutes±30 seconds, about 3minutes±30 seconds, about 4 minutes±30 seconds, about 5 minutes±30seconds, about 6 minutes±30 seconds, or about 7 minutes±30 seconds.

Provided in another embodiment is a method for extracting a nucleic acidfrom a cellular material in a sample comprising a bodily fluid or aninoculant derived therefrom, the method comprising the steps of (a)subjecting the sample to a first lysing process comprising mechanicallysis to cause disruption of a cellular membrane in the cellularmaterial; (b) subjecting the sample to a second lysing processcomprising at least one of physical lysis, chemical lysis, biologicallysis and any combination of two or more of these to produce a lysatecomposition comprising the nucleic acid; and (c) recovering the lysatecomposition from the sample, wherein the mechanical lysis may comprise acombination of centrifugation and puck lysing. In some embodiments, thepuck lysing may be magnetic puck lysing. In certain preferredembodiments, the combination of centrifugation and puck lysing may becarried out in a common lysis chamber, preferably centrifugation andpuck lysing may be carried out on a centrifugal disk.

Provided in another embodiment is a method for extracting a nucleic acidfrom a cellular material in a sample comprising a bodily fluid or aninoculant derived therefrom, the method comprising the steps of (a)subjecting the sample to a first lysing process comprising mechanicallysis to cause disruption of a cellular membrane in the cellularmaterial; (b) subjecting the sample to a second lysing processcomprising at least one of physical lysis, chemical lysis, biologicallysis and any combination of two or more of these to produce a lysatecomposition comprising the nucleic acid; and (c) recovering the lysatecomposition from the sample, wherein steps (a) and (b) may be carriedout concurrently.

Provided in another embodiment is a method for extracting a nucleic acidfrom a cellular material in a sample comprising a bodily fluid or aninoculant derived therefrom, the method comprising the steps of (a)subjecting the sample to a first lysing process comprising mechanicallysis to cause disruption of a cellular membrane in the cellularmaterial; (b) subjecting the sample to a second lysing processcomprising at least one of physical lysis, chemical lysis, biologicallysis and any combination of two or more of these to produce a lysatecomposition comprising the nucleic acid; and (c) recovering the lysatecomposition from the sample, wherein steps (a) and (b) may be carriedout sequentially. In certain preferred embodiments, step (b) may becarried out after commencement of disruption of the cellular membrane instep (a).

The methods disclose herein may comprise performing one or moremechanical lyses and one or more non-mechanical lyses.

In some embodiments, lysing the microorganism occurs prior todetermining the quantity of a nucleic acid molecule in a plurality ofinoculates.

In some embodiments, the methods disclosed herein comprise contactingthe neutralized cell lysate with a solution comprising streptavidin.

Nucleic Acid Molecule Quantification

In some embodiments, the methods disclosed herein comprise detecting thequantity of a nucleic acid molecule from a microorganism in a sample. Insome embodiments, the methods disclosed herein comprise comparing thequantity of a nucleic acid molecule in the antimicrobial agent-freeinoculate to the quantity of a nucleic acid molecule in theantimicrobial agent inoculate. In some embodiments, the nucleic acidmolecule is a deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or acombination thereof.

In some embodiments, the methods disclosed herein are a RiboResponse™method. In some embodiments, the RiboResponse™ method comprisesdetermining the quantity of an RNA molecule from the microorganism. Insome embodiments, the RNA is a mature RNA. In some embodiments, the RNAis a precursor RNA. In some embodiments, the RNA is a ribosomal RNA(rRNA). In some embodiments, the rRNA is a 16S RNA or 23S RNA. In someembodiments, the microorganism is a prokaryote. In some embodiments, theprokaryote is a Gram-negative bacterium. In some embodiments, theprokaryote is a Gram-positive bacterium. In some embodiments, themicroorganism is fungal (e.g., candida).

The RiboResponse™ platform is quantitative in that more bacteria wouldresult in more ribosomes and, hence, ribosomal RNA, resulting in ahigher detection signal when ribosomal RNA is detected. In someembodiments, the detected level of a nucleic acid molecule in each ofthe plurality of inoculates comprising an antimicrobial agent for eachantibiotic is compared to the control lacking an anti-microbial agent(ideal growth) and expressed as a percentage of the no antibioticcontrol. In some embodiments, resistant antibiotics have numbers closeto 100%, meaning they had a comparable level of growth to the noantibiotic control. In some embodiments, an inoculate with anantimicrobial agent to which a microorganism is susceptible antibioticswill have a nucleic acid molecule detection level lower than 100%, e.g.,90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%,20%, 15%, 10%, or 5%, or less.

In some embodiments, determining the quantity of a nucleic acid moleculein an inoculate allows for an estimation of bacterial density (orquantity) in the inoculate. In some embodiments, the density of bacteriain a sample (or a particular inoculate) is estimated by applying aformula that contains variables m (slope) and b (y-intercept) derivedfrom an empirically determined species-specific standard curve. Thebacterial assay signal is normalized by dividing it by the assaypositive control signal and multiplying the result by 1000. Wherey=log₁₀ (normalized assay signal), the formula that relates the assaysignal to CFU/mL is:

${\log_{10}( {{CFU}\text{/}{ml}} )} = \frac{( {y - b} )}{m}$

In some embodiments, a predicted CFU/mL value is multiplied by anadjustment factor F_(a) to provide an improved estimate of finalbacterial density, where F_(a) is based on comparing formula predictionsto observed inoculation results:

$F_{a} = {1 + {5.76 \times {e^{- 0.5}\lbrack \frac{{\log_{10}{CFU}\text{/}{ml}} - 7.23}{0.259} \rbrack}^{2}}}$

In some embodiments, the plurality of inoculates is serially diluted incell culture media prior to quantification of a nucleic acid molecule.

In some embodiments, determining the quantity of a nucleic acid moleculein a plurality of inoculates comprises a sandwich assay. In someembodiments, determining the quantity of a nucleic acid molecule in aplurality of inoculates comprises using an electrochemical sensorplatform. In some embodiments, determining the quantity of a nucleicacid molecule in a plurality of inoculates comprises using an ELISA. Insome embodiments, determining the quantity of a nucleic acid molecule ina plurality of inoculates comprises using a magnetic bead-baseddetection platform.

Antimicrobial Agent Susceptibility

Disclosed herein are methods for determining the susceptibility of amicroorganism to an antimicrobial agent. In some embodiments, themicroorganism is susceptible to the antimicrobial agent if the quantityof nucleic acid molecules of the microorganism in the antimicrobialagent-free inoculate is more than the quantity of nucleic acid moleculesof the microorganism in an inoculate comprising the microorganism andthe antimicrobial agent. In some embodiments, the microorganism is notsusceptible to the antimicrobial agent if the quantity of nucleic acidmolecules of the microorganism in the antimicrobial agent-free inoculateis nearly equal, equal, or less than the quantity of nucleic acidmolecules of the microorganism in an inoculate comprising themicroorganism and the antimicrobial agent.

Reports and Data Transmission

In some embodiments, the methods disclosed herein further comprisegenerating one or more reports. In some embodiments, the methodsdisclosed herein further comprise transmitting one or more reports. Insome embodiments, the report includes information on the susceptibilityof a microorganism to one or more antimicrobial agents or combinationsof antimicrobial agents. In some embodiments, the report providesrecommendations on a therapeutic regimen. In some embodiments, thereport provides recommendations on the dosage of an antimicrobial agent.

Detecting the Presence of Microorganisms in a Clinical Specimen

When using some of the methods described herein, the quantity of themicroorganism in the sample to be tested may be known. For example, asample may be prepared for the purpose of undergoing the AST techniquesdescribed herein, and one or more of its parameters may be prescribed aspart of the test procedure. This may be the case in research orlaboratory testing environments. In other circumstances, at least someof the properties of the sample that is to be subjected to the ASTanalysis are very likely to be unknown to those performing the test. Forexample, if a clinical specimen is taken from a patient for analysis,the nature of the microorganisms (if any) and their quantity within thesample may be unknown. Without at least some type of approximate or“good enough” estimation of the quantity of the microorganism (e.g.bacteria) in the sample it may be relatively more difficult to calibratethe inputs of a desired AST process. For example, it may be moredifficult to determine if a sample ought to be diluted, and if so bywhat extent, and to select an appropriate dosage(s) of the one or moreantimicrobial agents that may be used in the process. That is,quantification of bacterial density may, for example, be useful indetermining the correct inoculation of a clinical specimen into growthmedium for the AST. It may also be useful in determining if an infectionis present or not.

To help reduce this uncertainty, particularly when desiring to testsamples of unknown contents, it may, in some circumstances, be desirableto quantify the bacterial density in a clinical specimen prior toconducting the AST.

Some examples of suitable methods for quantify the bacterial density ina clinical specimen have been disclosed in provisional patentapplication Ser. No. 62/671,380, filed May 14, 2018, the contents ofwhich are hereby incorporated by reference herein in its entirety.

In some embodiments, the methods disclosed herein further comprise stepsof quantifying bacterial density in a clinical specimen. Suchquantification is based on the following Translation Function:

[rRNA]=f(z)·cfu/ml

where z is the number of rRNA copies per cell, [rRNA] is the bacterialrRNA concentration, and CFU/mL is the bacterial density in abacteria-containing specimen.

As indicated by the above equation, the number of rRNA copies per cell(z) may be a linear function, which may be at least partially dependenton bacterial concentration. FIG. 22 shows an equation that relates rRNAcopies per cell to bacterial concentration in urine specimens.

In accordance with one aspect of the teachings described herein, amethod of in a urine specimen of a patient with a urinary tractinfection (UTI) is described. FIG. 20 is a flowchart illustrating oneembodiment of this method.

FIG. 20 sets out one example of a method 100 of estimating the bacterialdensity in a clinical specimen. This method includes a first step 102 ofobtaining a clinical specimen. In most embodiments of the method, theclinical specimen is believed to contain at least one species ofbacteria in a clinically relevant amount, and may be suspected ofcontaining two or more species of bacteria in a clinically relevantamount. In the illustrated example, the clinical specimen is a urinespecimen obtained from a patient that is complaining of symptomsconsistent with a urinary tract infection and the specimen is suspectedof containing at least a clinically relevant amount of E. coli.

Once the specimen suspected of containing a clinically relevant amountof bacteria is obtained, in a second step 104, the rRNA of the bacteriain the specimen is processed to obtain an rRNA signal. At least onepositive control and at least one negative control are included in step104.

In some embodiments of the invention, the time it takes from when aclinical specimen is obtained (i.e. step 102) to when the rRNA of atleast one bacterial species in the clinical specimen has been processed(i.e. step 104) is less than four (4) hours. In some preferredembodiments, the time it takes from when a clinical specimen is obtained(i.e. step 102) to when the rRNA of at least one bacterial species inthe clinical specimen has been processed (i.e. step 104) is less than 3hours; less than 2 hours; less than 1 hour; less than 30 minutes; orless than 15 minutes.

The rRNA signal obtained from step 104 may then be used to determine therRNA concentration of the bacteria in the specimen, preferablyautomatically when using a suitable system (i.e. without requiringintervention from a skilled technician). A determination of rRNAconcentration may be based on a linear log-log correlation between theassay signal and the concentration of the rRNA analyte. Therefore, in anext step 106, the log of the rRNA signal from step 104 may becalculated to give the rRNA signal_(LOG).

In a next step 108, the log of the negative control signal from step 104is subtracted from both the rRNA signal_(LOG) from step 106 and the logof the positive control signal from step 104. The resulting rRNAsignal_(LOG) is then compared with the resulting positive controlsignal_(LOG) to normalize the signal intensity of the rRNA signal_(LOG)and determine the rRNA concentration of bacteria in the clinicalspecimen (units=pM,c Log 10).

In a next step 110, the rRNA concentration from step 108 may be inputtedinto a predetermined translation function to estimate the bacterialdensity value_(LOG) in the clinical sample (units=CFU/ml, Log 10).

In a next step 112, the inverse log of the bacterial density value_(LOG)from step 110 may be calculated to estimate the bacterial density valueof the clinical specimen (units=CFU/ml).

RNA Quantification (Step 104)

Determining the concentration of rRNA may be done using any suitablemethod, including those described herein. One example of a suitablemethod may include the steps of: 1) Lysis to release rRNA 128; 2)Neutralization 130; 3) Hybridization of target rRNA with a capture probeand detector probe 132; and 4) Detection of capture probe—targetrRNA—detector probe complexes 134.

Optionally, the method of determining the concentration of the rRNA maybe performed at least partially, and preferably completely,automatically using a suitable apparatus.

In the illustrated example, a MagPix (Luminex) magnetic bead assay isused to measure the E. coli rRNA concentration in fresh urine specimensfrom a patient with UTI.

Lysis (Step 128)

Optionally, the lysing step 128 may include at least one of chemicallysing, mechanical lysing, and/or a combination thereof. In a preferredembodiment, lysis 128 may include both chemical and mechanical lysingoperations, as described above and in PCT/US18/45211, which isincorporated herein by reference herein in its entirety.

Neutralization (Step 130)

The neutralization step 130 can be performed using any known or unknownmethod.

In the illustrated example, samples are lysed with one-half volume of 1MNaOH. This lysate is neutralized with an equal volume of 1Msodium-potassium phosphate buffer, pH 6.4.

Hybridization (Step 132)

Preferably, a species-specific signal can be provided for each type oftarget bacteria that is expected to be present in the clinical specimen.By using a species-specific signal, the signal of rRNA from differenttypes of bacteria in mixed specimens may be individuallyobserved/counted and/or only signals from the desired, targeted bacteriamay be counted. This may help facilitate the quantification of two ormore different target bacteria within a common clinical specimen, andmay allow the concentrations of two or more target bacterial rRNAconcentrations to be measured generally simultaneously.

This may be advantageous when analyzing certain types of clinicalspecimens, such as urine specimens, which may tend to include a varietyof different bacteria in generally unknown quantities at the beginningof the analysis process. By using species-specific signal probes, themethods described herein could be used to independently determine aquantity of rRNA from two or more specific bacterial species in theclinical specimen, input those values into respective, pre-determinedtransfer functions and calculate respective rRNA concentration valuesfor each bacterial species. These results can then be used to provideoutputs and/or as inputs in other method steps on a species-specificbasis. For example, the methods may indicate a bacterial density valuefor E. coli that is above an E. coli pre-determined treatment threshold,while a bacterial density value for K. pneumoniae is below itsrespective pre-determined treatment threshold. This may be used toinitiate further treatment or diagnoses methods regarding E. coli, whilenot initiating analogous steps for K. pneumoniae. Alternatively, if bothbacterial density values are above their respective pre-determinedtreatment thresholds, a different, suitable treatment protocol may beselected or followed.

Detection (Step 134)

A variety of platforms can be used for detection 134, including but notlimited to excitation and imaging of fluorescent-tagged detector probes,bioluminescence using luciferase-type enzymes, and amperometric currentusing an electrochemical sensor. In the illustrated example,fluorescent-tagged detector probes are used for detection.

During detection 134, at least one positive control and at least onenegative control are included. In the illustrated example, a syntheticoligonucleotide with the same sequence as the target rRNA is included asa positive control and a sample without rRNA or bacteria is included asa negative control.

Translation Function

The translation function used in step 110 is preferably selected fromamongst one or more pre-determined translation functions. Suitabletranslation functions may be determined using any suitable technique,including those described herein. Optionally, more than one translationfunction may be determined and may be stored or otherwise recorded in atranslation function table. For example, different translation functionsmay be developed for different species of bacteria that may be expectedto be present in an incoming clinical specimen. That is, one translationfunction may be used to correlate the rRNA concentration and CFU/mL ofE. coli in a given specimen, while a different translation function maybe used to correlate the concentration of rRNA and CFU/mL of K.pneumoniae. Some translation functions may be better suited for use witha given type of bacteria.

Each translation function may take as an input a value that is based onthe species-specific rRNA concentration in the specimen. For example, atranslation function derived for E. coli may take as its input a valuecorresponding to the rRNA concentration of E. coli in the specimen,whereas a translation function for K. pneumoniae may take as its input avalue corresponding to the rRNA concentration of K. pneumoniae in thespecimen.

If more than one translation function has been determined, the methodsand/or systems described herein may include the steps of selecting onetranslation function, from the two or more translation functionsavailable, as being most appropriate for use with a given clinicalspecimen. The selection of a given translation function may be based ona variety of factors, including user inputs/selections, the expectedtypes of bacteria, the type of specimen, ambient temperature, and samplestorage time.

In a preferred embodiment, a translation function is derived from abacterial species-specific standard curve. To derive a bacterialspecies-specific standard curve, rRNA concentrations of a specificbacteria may be measured in a group of clinical specimens of the sametype (e.g. a group of urine specimens). Species-specific bacterialdensities may then be determined on the same specimens using any knownmethod. This relationship may then be plotted on a graph, with rRNAconcentration (pM, Log 10) on one axis and CFU/mL (Log 10) on the otheraxis to determine the correlation between rRNA concentration andbacterial density. The resulting relationship between these twovariables may define a translation function.

The number of specimens required to derive a bacterial species-specificstandard curve may depend on such factors as the type of specimen andthe species of bacteria being analyzed. The number of specimens requiredto accurately define a relationship between rRNA concentration andbacterial density may be determined using known statistical methods.

In the illustrated example, in a first step 136, a MagPix (Luminex)magnetic bead assay is used to measure E. coli rRNA concentrations infresh urine specimens from 25 patients with UTI, as according to steps102-108. In a next step 138, the bacterial density of E. coli in eachspecimen is determined with plate counts. In a next step 140, the log ofeach bacterial density from step 138 is calculated for each specimen toobtain the bacterial density_(LOG), which, in a next step 142, isplotted on a scatterplot against the rRNA concentration from step 136.From this scatterplot, the correlation between rRNA concentration andbacterial density is determined. FIG. 21 illustrates the correlationbetween E. coli rRNA concentration and density of E. coli for urinespecimens from 25 patients with E. coli urinary tract infection.

In the illustrated example, the slope of the resulting regression linemay be used as the translation function to estimate the E. colibacterial density value (CFU/ml) in a urine specimen. More specifically,the linear equation of the resulting regression line, as represented bythe general formula y=mx+b, may be used to estimate the bacterialdensity value (CFU/ml) of E. coli in a clinical specimen, wherein x isthe rRNA concentration of E. coli in a clinical specimen (pM, Log 10)and y is the bacterial density value of E. coli in the clinical specimen(CFU/ml, Log 10).

In the illustrated example, the linear equation of the resultingregression line, and therefore the translation function, is y=1.79x+3.5,as seen in FIG. 21. Therefore, in the illustrated example, thetranslation function for E. coli was empirically determined to bey=1.79x+3.5, where y is CFU/mL (log 10) and x is the rRNA concentration(pM, Log 10) value for the tested clinical specimen. The bacterialdensity value in units of CFU/mL can then be obtained by taking theinverse log of y. In other words, the bacterial density value for E.coli can be described as:

bacterial density value=antilog (1.79x+3.5)

While in this example the x coefficient is presented with threesignificant digits, other examples of the translation function may haveonly a single decimal point or may be otherwise rounded while stillproviding a sufficiently accurate output for the bacterial density valueon which to base clinical decisions.

Bacterial Density Value

Optionally, the bacterial density value (from step 112) can be providedto a user, for example via any suitable type of user display apparatus,such as a screen, print-out, email, text message, graphic, or the like.This information may then be used for any suitable purpose, including,for example, reporting and/or regulatory compliance.

In some embodiments, the bacterial density value may be used as an inputor otherwise implicated in other sorts of methods. For example, in oneembodiment, the bacterial density value may be used to determine thelikelihood of infection. In another embodiment, the bacterial densityvalue may be used as one of the inputs in a method or process that is tobe performed on the clinical specimen. In another embodiment, thebacterial density value may be used as a predictor of wound healingand/or acceptance of grafts.

Screening for Infection

Quantification of bacterial density may be useful in testing clinicalspecimens for the presence of bacteria above a certain predeterminedcutoff or threshold. Bacterial densities above the cutoff may beconsidered positive and indicate the presence of infection; bacterialdensities below the cutoff may be considered negative and may indicatesuch factors as contamination of the specimen during collection oroutgrowth of contaminants during storage or transport.

In the illustrated example, at step 144, a false negative rate of <5% isdetermined to be sufficient to assess the likelihood of infection in aclinical specimen. This means that the cutoff for the assessment ofinfection is set to 2 standard deviations above background, meaning thatif the bacterial density value of a specimen is greater than or equal to2 standard deviations above background, there is a likelihood ofinfection. Conversely, if the bacterial density value of a specimen isless than 2 standard deviations above background, there is not alikelihood of infection.

In the illustrated example, the likelihood of infection in a clinicalspecimen is assessed in steps 114-118. As a first step 114, thebacterial density value of E. coli in a urine specimen (from step 112)is compared with the predetermined infection threshold of 2 standarddeviations above background (from step 144). If the bacterial densityvalue from step 112 is greater than or equal to the infection threshold(i.e. ≥2 standard deviations above background), a positive outputindicating the likelihood of infection is produced, as seen at step 116.Alternatively, if the bacterial density value from step 112 is less thanthe infection threshold (i.e. <2 standard deviations above background),a negative output indicating that infection is not likely is produced,as seen at step 118.

AST Inoculation Concentration

Quantification of bacterial density may be useful in determining thecorrect inoculation of a clinical specimen into growth medium for adirect from specimen phenotypic AST. Providing a bacterial density valuethat is within an acceptable resolution for clinical analysis may helpdetermine an appropriate dosage of an inoculation agent to be used witha given clinical specimen to help provide a desired or targetinoculation concentration in the clinical specimen. Utilizing thebacterial density value as a factor to help determine the dosage of theinoculation may help reduce the likelihood of over or under-diluting agiven clinical specimen during further processing.

For example, in one embodiment, the target inoculation concentration ofthe AST may be 5×10⁵ CFU/ml. Inoculation concentrations up to 5×10⁶CFU/mL may provide an accurate AST result, whereas inoculationconcentrations greater than 5×10⁶ CFU/mL may limit growth, therebypossibly reducing accuracy of AST results.

In the illustrated example, the determination of the AST inoculationconcentration of the clinical specimen is set out in steps 120-126. As afirst step, the bacterial density value from step 112 is compared to thepredetermined desired target inoculation concentration for AST. If thebacterial density value from step 112 is greater than the desired targetinoculation concentration, step 122 is engaged, in which the dilutionfactor required to dilute the bacterial density value of the specimen towithin the desired target inoculation concentration range is determined.Based on the calculated dilution factor from step 122, growth medium isadded to dilute the specimen to within the desired target range, as perstep 124. The specimen can then be inoculated into growth medium for theAST, as per step 126.

On the other hand, if the bacterial density value from step 112 is lessthan or equal to the desired target inoculation concentration, thespecimen may be inoculated into growth medium for the AST withoutdilution. In other words, steps 122-124 may be by-passed and the userwould go immediately to step 126.

Automation

Preferably, some or all of the steps in the methods can be automatedusing suitable equipment and do not require a skilled laboratorytechnician or the like to process the specimens and/or interpret theresults. In some embodiments described herein, the inputs for theanalysis method is a generally “fresh”, unmodified specimen obtaineddirectly from a subject and the output of the method is an answer thatis usable and/or understandable by a lay operator (i.e. not a skilledlab technician). For example, the output may be in the form of a numberthat represents the concentration of the target bacteria within thespecimen.

EXPERIMENTAL EXAMPLES

Embodiments of the present invention will now be illustrated withreference to the following examples which should not be used to construeor limit the scope of the present invention.

A. Determining the Susceptibility of Bacteria to Antibiotics Using aRiboresponse™ Method

In this example, the materials and methods for performing aRiboResponse™ method for determining the susceptibility of bacteria to aplurality of antibiotics are provided.

Materials

-   -   1. Detector probe buffer: Mixture of detector probes (100 nM) in        1 M Phosphate Buffer pH 6.4.    -   2. Bead plate: 96-well plate containing Luminex MTAG beads        functionalized with capture probes.    -   3. Positive control: 100 pM synthetic target in 1 M Phosphate        Buffer pH 6.4.    -   4. AST plate: 96-well plate containing 180 μl of Cation-adjusted        Mueller Hinton (MH2) broth per well, containing the working        concentration of the appropriate antibiotic in the appropriate        wells. To this plate is applied a 96-well plate sticker to        prevent cross-contamination and evaporation.    -   5. Lysis plate: 96-well plate containing 25 μl of 1M NaOH per        well.    -   6. 1×Tm HB=0.1 M Tris pH 8.0, 0.2 M NaCl, 0.08% Triton X-100    -   7. 1 M NaOH    -   8. Streptavidin-phycoerythrin conjugate

Equipment

-   -   1. Shaker Incubator    -   2. Biotek 405TS Plate Washer    -   3. Luminex MagPix Assay System

Set 1 of Antibiotic Concentrations (working concentrations):

-   -   1. Gentamicin 4 μg/ml    -   2. Ciprofloxacin 4 μg/ml    -   3. Cefazolin 64 μg/ml    -   4. Ceftriaxone 32 μg/ml    -   5. Cefepime 64 μg/ml    -   6. Ampicillin 512 μg/ml    -   7. Imipenem 4 μg/ml    -   8. Trimethoprim 8 μg/ml and Sulfamethoxazole 152 μg/ml    -   9. Amikacin 64 μg/ml    -   10. Nitrofurantoin 16 μg/ml    -   11. Fosfomycin 64 μg/ml    -   12. Piperacillin 16 μg/ml and Tazobactam 4 μg/ml    -   13. Amoxicillin 32 and Clavulanate 16

Set 2 of Antibiotic Concentrations (working concentrations):

-   -   1. Gentamicin 4 μg/ml and 2 μg/ml    -   2. Ciprofloxacin 4 μg/ml    -   3. Cefazolin 64 μg/ml    -   4. Ceftriaxone 32 μg/ml    -   5. Cefepime 64 μg/ml and 32 μg/ml    -   6. Ampicillin 128 μg/ml    -   7. Trimethoprim 4 μg/mL and Sulfamethoxazole 76 μg/ml    -   8. Amikacin 32 μg/ml, 16 μg/ml and 8 μg/ml    -   9. Nitrofurantoin 16 μg/ml    -   10. Fosfomycin 64 μg/ml    -   11. Amoxicillin 64 μg/mL and Clavulanate 32 μg/ml; Amoxicillin        32 μg/mL and Clavulanate 16 μg/ml; Amoxicillin 16 μg/mL and        Clavulanate 8 μg/ml    -   12. Etrapenem 4 μg/ml and 2 μg/ml    -   13. Meropenem 4 μg/ml and 2 μg/ml

Method 1: RiboResponse™ Method Using a Microtiter Plate

-   -   1. The AST plate was prewarmed and aerated by shaking in the        37° C. shaker incubator at 400 rpm.    -   2. The specimen was adjusted to a concentration of −5×10⁶        cfu/ml.    -   3. The wells of the 96-well AST plate were inoculated by adding        20 μl to the 180 μl in the well to yield 5×10⁵ CFU/ml.        Uninoculated wells were included for negative and positive        controls.    -   4. After inoculation, a 50 μl sample was transferred from the 0        min No Abx well to the corresponding well in the lysis plate        containing 25 μl 1 M NaOH and mixed by pipetting.    -   5. The inoculated 96-well plate was placed in the 37° C. shaking        incubator at 400 rpm for 90-120 minutes.    -   6. After 5 minutes of incubation at room temperature, 75 μl of        detector probe buffer was added to neutralize the lysate and        mixed by pipetting.    -   7. Steps 5 and 6 were repeated for the other wells in the AST        and Lysis plates at the end of the 90-120 minute incubation        period.    -   8. The negative control was neutralized in the same way and 100        pM synthetic target in detector probe buffer was used for the        positive control.    -   9. The bead plate was shaken using the 2 minute fast shaking        cycle on the Biotek Plate washer    -   10. The beads were washed in the Biotek plate washer using the        Biotek Bead Washing Protocol below.    -   11. The multichannel pipettor was used to add 25 μl of the bead        capture probe mixture from the 96-well bead plate to each well        in the lysis plate.    -   12. The plate was shaken (without magnet) for 15 minutes on the        variable setting with the Biotek plate washer.    -   13. The beads were washed in the Biotek plate washer using the        Biotek Bead Washing Protocol below.    -   14. While the plate was washing, 2 μl of 1 mg/mL Streptavidin-PE        stock was added to 1000 1×Tm HB to yield 2 μg/ml.    -   15. After the plate has finished washing, 75 μl 2 μg/mL        Streptavidin-PE was added to the appropriate wells.    -   16. The plate was shaken on variable speed with the Biotek plate        washer for 1 minute.    -   17. The beads were washed with the Biotek plate washer following        the protocol listed below.    -   18. The beads were measured in the Luminex MagPix instrument.

Method 2: Riboresponse™ Method Using a Centrifugal Disc

The RiboResponse™ method using a centrifugal disc is similar to Method 1(above), except that the 90-120-minute incubation was performed inincubation chambers of a centrifugal disc. As shown in FIG. 1, growth ina rotating centrifugal disc was significantly faster than growth in ashaking centrifugal disc or shaking 96-well plate. Accelerated growthenables faster separation of susceptible and resistant bacteria. FIG. 24is another example, illustrating enhanced results when incubation wasconducted on a centrifugal disc.

Biotek Bead Washing Protocol (using 96-well plate magnet)

-   -   1. Shake on medium for 30 seconds    -   2. Soak for 30 seconds    -   3. Aspirate    -   4. Dispense 200 μl of 1×Tm HB per well    -   5. Shake on medium for 30 seconds    -   6. Soak for 30 seconds    -   7. Aspirate    -   8. Dispense 200 μl of 1×Tm HB per well    -   9. Shake on medium for 30 seconds    -   10. Soak for 30 seconds    -   11. Aspirate    -   12. FINAL WASH ONLY: Dispense 50 μl

Biotek 96 well plate washer settings:

-   -   Aspirate options—Z=43 (5.46 mm above carrier), X=30 (1.37 mm        right of center)    -   Dispense options—Z=130 (16.52 mm above carrier), X=0    -   Slow mixing→7 Hz (420 rpm)    -   Medium mixing→13 Hz (780 rpm)    -   Fast mixing→19 Hz (1140 rpm)    -   Variable mixing→repeated cycle of (slow, medium, and fast        mixing)×∞, cycles are ˜1.5 seconds each

B. Cell Lysis Example 1. Cell Lysis Using Mechanical and Non-MechanicalLysis

In this Example, the materials and methods for lysing bacteria (e.g.,Staphylococcus aureus) using mechanical lysis (OmniLyse® or centrifugaldisk) and non-mechanical lysis (NaOH) are provided.

Materials

The following materials were used:

-   -   1. OmniLyse® Lysis Kit. Available from ClaremontBio.com:        http://www.claremontbio.com/OmniLyse_Cell_Lysis_Kits_s/56.htm;    -   2. 1.7 mL microcentrifuge tubes;    -   3. mixture of identification (ID) detector probes (100 nM) in 1        M phosphate buffer pH 6.4;    -   4. 96-well plate containing Luminex MTAG beads functionalized        with capture probes;    -   5. 1×Tm HB=0.1 M Tris pH 8.0, 0.2 M NaCl, 0.08% Triton X-100;    -   6. 1 M NaOH; and    -   7. Streptavidin-phycoerythrin conjugate.

Equipment

The following equipment was used:

-   -   1. Shaker Incubator;    -   2. Biotek 405TS Plate Washer; and    -   3. Luminex MagPix Assay System.

Method 1: OmniLyse® and NaOH

The following methodology were used:

-   -   1. The OmniLyse® cartridges were pre-wetted by filling the        cartridge with filter-sterilized superwater, and emptying with        the syringe plunger. This step was repeated one additional time.        One OmniLyse® cartridge was needed for each specimen and        control.    -   2. 40 μl of 1 M NaOH was added to 1.7 mL microcentrifuge tubes.        2 extra tubes were included for negative and positive controls.    -   3. 80 μl of specimen was added to a microcentrifuge tube that        contained 40 μl 1 M NaOH and mixed by pipetting.    -   4. The syringe plunger was used to draw 120 μl of specimen+NaOH        from the sample tube into the OmniLyse® cartridge. The OmniLyse®        cartridge was turned on for 1 minute.    -   5. After OmniLyse® treatment, the plunger was used to dispense        up to 120 μl of lysate into a tube and incubated at room        temperature to complete the 5 minutes of exposure to NaOH.    -   6. The lysates were neutralized by adding 100 μl of ID detector        probe mixture to each tube and mixed by pipetting.    -   7. 190 μl of neutralized lysate was added to wells in the        96-well ID plate. Negative and positive control lysates were        also added.    -   8. The plate was shaken (without magnet) for 15 minutes on the        variable setting with the Biotek plate washer.    -   9. The beads were washed in the Biotek plate washer using the        Biotek Bead Washing Protocol below.    -   10. While the plate was washing, 2 μl of 1 mg/mL Streptavidin-PE        stock was added to 1000 μl 1×Tm HB to yield 2 μg/ml.    -   11. After the plate was finished washing, 75 μl of 2 μg/mL        Streptavidin-PE was added to the appropriate wells.    -   12. The plate was shaken on variable speed with the Biotek plate        washer for 1 minute.    -   13. The beads were washed with the Biotek plate washer following        the protocol listed below.    -   14. The beads were then measured in the Luminex MagPix        instrument.

Method 2: Centrifugal Disk and NaOH

The method for performing mechanical lysis using a centrifugal disk issimilar to Method 1 described above, except that the OmniLyse in step 4of Method 1 was replaced by a centrifugal disk containing a lysischamber containing zirconium beads and a stainless-steel lysing puck(see FIG. 9). 120 μl of specimen and NaOH from step 3 of Method 1 wasplaced in the CD lysis chamber and the centrifugal disc was rotated at100 rpm for 5 minutes. As the centrifugal disc rotated on the spinplatform, magnets below the disc caused the stainless-steel lysing pucksto move back and forth in the lysis chamber, which when combined withzirconium beads provided grinding action.

Biotek Bead Washing Protocol (using 96-well plate magnet):

-   -   1. Shake on medium for 30 seconds    -   2. Soak for 30 seconds    -   3. Aspirate    -   4. Dispense 200 μl of 1×Tm HB per well    -   5. Shake on medium for 30 seconds    -   6. Soak for 30 seconds    -   7. Aspirate    -   8. Dispense 200 μl of 1×Tm HB per well    -   9. Shake on medium for 30 seconds    -   10. Soak for 30 seconds    -   11. Aspirate    -   12. FINAL WASH ONLY: Dispense 50 μl

Biotek 97 well plate washer settings:

-   -   1. Aspirate options—Z=43 (5.46 mm above carrier), X=30 (1.37 mm        right of center)    -   2. Dispense options—Z=130 (16.52 mm above carrier), X=0    -   3. Slow mixing→7 Hz (420 rpm)    -   4. Medium mixing→13 Hz (780 rpm)    -   5. Fast mixing was performed at 19 Hz (1140 rpm).

Variable mixing comprised repeated cycles of slow, medium, and fastmixing at approximately 1.5 seconds each.

As shown in FIG. 10, the combination of mechanical lysis andnon-mechanical lysis of Staphylococcus areus resulted in more efficientlysis than non-mechanical lysis with NaOH alone. FIG. 10 shows that at50, 100 and 200 revolutions per minute (RPM), mechanical lysis with acentrifugal disk in combination with non-mechanical lysis using NaOH(first column) and mechanical lysis with OmniLyse® in combination withnon-mechanical lysis using NaOH (third column) resulted in moreefficient lysis compared to chemical lysis using NaOH alone (secondcolumn). The efficacy of the cell lysis was measured by detecting thequantity of rRNA released from identical samples.

As shown in FIG. 11, mechanical lysis with a centrifugal disk incombination with non-mechanical lysis using NaOH (first column) andmechanical lysis with OmniLyse® in combination with non-mechanical lysisusing NaOH (third column) resulted in more efficient lysis for a broadvariety of Gram-positive bacteria compared to chemical lysis using NaOHalone (second column). The efficacy of the cell lysis was measured bydetecting the quantity of rRNA released from identical samples.

Example 2. Mechanical Lysis and Non-Mechanical Lysis of Gram-PositiveBacteria Results in More Efficient Detection of rRNA as Compared to aCombination of Enzymatic Lysis, Detergent Lysis and Chemical Lysis

In this Example, using the relevant materials and methodology describedin Example 1, Gram-positive bacteria were lysed using a two-step lysisusing either (a) Step 1: enzymatic lysis and detergent lysis, and Step2: chemical lysis (e.g., Step 1: Triton X-100 and lysozyme, and Step 2:NaOH); or (b) Step 1: mechanical lysis and Step 2: chemical lysis (e.g.,Step 1: OmniLyse® and Step 2: NaOH), followed by detection of rRNA usinga Luminex® instrument.

As shown in FIG. 12, the detection of rRNA was greatly increasedfollowing mechanical lysis using OmniLyse® in combination with chemicallysis using NaOH (first column) as compared to the detection of rRNAfollowing enzymatic lysis using lysozyme and detergent lysis usingTriton X-100 in combination with chemical lysis using NaOH.

As shown in FIG. 13, mechanical lysis using OmniLyse® in combinationwith chemical lysis using NaOH (first column) resulted in improveddetection of rRNA from a broad variety of Gram-positive bacteria (e.g.,Staphylococcus aureus, Staphylococcus lugdunensis, Enterococcusfaecalis, Streptococcus pyogenes, and Streptococcus Agalactiae) comparedto enzymatic lysis using lysozyme and detergent lysis using Triton X-100in combination with chemical lysis using NaOH.

These results demonstrate that the first step of enzyme plus detergentfollowed by NaOH treatment results in less efficient detection of rRNAfrom Gram-positive cells than the combination of mechanical lysis plusNaOH.

Example 3. Impact of the Duration of Mechanical Lysis and Concentrationof NaOH on rRNA Detection

In this Example, using the relevant materials and methodology describedin Example 1, the impact of the duration of mechanical lysis andconcentration of NaOH on rRNA detection from Staphylococcus aureus wasinvestigated. In the first step, bacteria were lysed for 1, 2, 3, 4, or5 minutes using OmniLyse® and then chemically lysed using 2M NaOH or 3MNaOH for a duration of 5 minutes. As shown in FIG. 14, an optimal signalwas achieved with mechanical lysis for 1 minute followed by chemicallysis using 3M NaOH.

A separate experiment was performed to determine the optimal duration ofNaOH treatment following a 1-minute mechanical lysis (OmniLyse®). Forall NaOH concentrations, the optimal duration of NaOH treatment wasfound to be 5 minutes (FIG. 15).

Example 4. Efficacy of Various Concentrations of Lysozyme Lysis Bufferon Gram-Positive Isolates

In step one of this example, the impact of biological (enzymatic in thiscase) lysis at different concentrations was investigated and compared toa combination of mechanical and alkaline lysis. During this experiment,a series of Gram-positive bacteria were lysed using differentconcentrations of lysozyme enzyme solution, either with or without theaddition of 1-minute mechanical lysis (OmniLyse®). Following lysis, thecell lysate was contacted with specific capture probes and detectorprobes, using the relevant materials and methodology described inExample 1, to detect one or more nucleotide sequences in the celllysate.

In step two, a separate experiment was performed, using the relevantmaterials and methodology described in Example 1, where Gram-positivebacteria were subjected to NaOH treatment following 1-minute mechanicallysis (OmniLyse®). The results for step one and step two were comparedas shown in FIG. 16.

Experimental Materials

The following materials were used:

-   -   1. OmniLyse® Lysis Kit. Available from ClaremontBio.com:        http://www.claremontbio.com/OmniLyse_Cell_Lysis_Kits_s/56.htm;    -   2. Bacteria samples including: MSSA 15-21-05; Staph Lugdunensis        ATCC; E. faecalis 07-09-53; Strep. pyogenes 15-21-26; and Strep.        agalactiae 07-09-45    -   3. Lysis buffer including:        -   (a) Lysozyme @ 1 mg/mL, Triton X-100 @ 0.1%, in H₂0        -   (b) Lysozyme @ 5 mg/mL, Triton X-100 @ 0.5%, in H₂0        -   (c) Lysozyme @ 10 mg/mL, Triton X-100 @ 0.5%, in H₂0        -   (d) Lysozyme @ 50 mg/mL, Triton X-100 @ 0.5%, in H₂0        -   (e) Lysozyme @ 1 mg/mL, Triton X-100 @ 0.1%, in 20 mM            Tris-HCl 2 mM EDTA pH 8.0        -   (f) Lysozyme @ 5 mg/mL, Triton X-100 @ 0.5%, in 20 mM            Tris-HCl 2 mM EDTA pH 8.0        -   (g) Lysozyme @ 10 mg/mL, Triton X-100 @ 0.5%, in 20 mM            Tris-HCl 2 mM EDTA pH 8.0        -   (h) Lysozyme @ 50 mg/mL, Triton X-100 @ 0.5%, in 20 mM            Tris-HCl 2 mM EDTA pH 8.0    -   4. 96-well plate containing Luminex MTAG beads functionalized        with capture probes; and    -   5. 1 M NaOH.

Experimental Methods

The following experimental variables were used for the Lysozyme BufferSet-Up:

The Lysozyme Buffers were made the same for every concentration,including:

-   -   a. 40 uL Bacteria+10 uL Enzymatic Lysis Buffer (5 min @ room        temperature)    -   b. 25 uL 1M NaOH (5 min)    -   c. 75 uL 1M Phosphate Buffer

Results

As shown in FIG. 8, the best enzymatic lysis condition used 50 mg/mLLysozyme and 0.5% Triton X-100—i.e., 3(d) and 3(h) above.

Example 5. Testing Relationship Between Strength of NaOH and Timing ofOmniLyse® Experimental Methods

In this example, two experiments were performed. In the firstexperiment, using the relevant materials and methodology described inExample 1, the relationship between strength of NaOH and timing ofOmnilyse® was investigated. In the first step, samples of Gram-positivebacteria (Staphylococcus aureus) were lysed for 1, 2, 3, 4, or 5 minutesusing OmniLyse® and then chemically lysed using 1M NaOH for 5 minutesafter OmniLyse® treatment. Results from this lysis were compared toenzymatic lysis as a control (See FIG. 17A)

In a second experiment, bacteria lysis of Gram-positive bacteria(Staphylococcus aureus) was performed with OmniLyse® for 2, 3.5 or 5minutes with 1M, 2M or 3M NaOH (See FIG. 17B).

Results

As shown in FIGS. 17A and 17B, the combination of mechanical andnon-mechanical lysis has proven to be effective in lysis ofGram-positive bacteria. The highest signal was found using 3M NaOH for 5minutes, 3M for 3.5 minutes and 2M for 5 minutes.

Example 6. Testing Combination Lysis Methods on Eukaryotic Fungal Cells(Candida Albicans)

In this example, using the relevant materials and methodology describedin Example 1, the effectiveness of different lysis methods was tested ondifferent cell types, including Gram-negative cells, Gram-positive cellsand eukaryotic fungal cells.

Experimental Materials

-   -   1. The following bacterial samples were used:        -   a. 10 Gram-negative, including E. coli, P. mirabilis, K            pneumoniae, K oxytoca, E. hormaechei, E. aerogenes, E.            cloacae, P. aeruginosa, C. freundii, and S. marcescens        -   b. 9 Gram-positive organisms, including S. aureus, S.            lugdunensis, E. faecalis, E. faecium, S. agalactiae, S.            pneumoniae, S. viridans, and S. pyogenes        -   c. 1 yeast, C. albicans    -   2. All bacteria were grown in MH2+5% LAKED horse blood+1 ug/mL        RnaseA    -   3. C. albicans was grown in RPMI overnight

Experimental Methods

For Gram-negative cells, alkaline lysis alone was used. ForGram-positive cells, a combination of alkaline lysis with OmniLyse®mechanical lysis was used. For eukaryotic fungal cells both alkalinelysis alone and a combination of alkaline lysis with OmniLyse®mechanical lysis were tested and compared. When the combination wasused, alkaline (chemical) lysis with 1M NaOH was performed for 5 minutesand Omnilyse® (mechanical) was performed for the first 2 minutes of the5 minutes alkaline (1M NaOH) lysis. Results for probe specificityfollowing the lysis of each cell type are shown in FIG. 18.

Results

As shown in FIG. 10, higher signals were obtained with the combinationof chemical and mechanical lysis as detected with eumicrobial (EU) orcandida (CN or CN-Help) probes.

Example 7. Comparison of Buffers for Neutralizing Lysate

Experimental Methods

In this experiment, cell lysate samples were neutralized by contactingthe samples with a buffer solution. During this experiment a series ofdifferent buffers were used, including: 1M Phosphate buffer (PB); 1MPB+1M NaCl; 1M Citrate buffer (CB); and 1M CB+1M NaCl and their abilityto neutralize NaOH in the lysate was compared. See FIG. 11.

Results

As shown in FIG. 19, when compared to an equal molarity strength ofCitrate buffer, the phosphate buffer was much better at neutralizing thelysate.

Antimicrobial Agents

The experiments discussed above have determined the followingpredetermined concentrations, when the desired inoculation period is90-120 minutes across a wide range of gram negative pathogens:

Gentamicin

In some embodiments, the at least one antimicrobial agent includesgentamicin. In some embodiments, the predetermined concentration ofgentamicin is equal to the CLSI MIC susceptible breakpoint. In someembodiments, the predetermined concentration of gentamicin is less thanthe CLSI MIC susceptible breakpoint.

In some embodiments, the predetermined concentration of gentamicin is atleast 2 μg/mL. In some embodiments, the predetermined concentration ofgentamicin is at least 4 μg/mL. In some embodiments, the predeterminedconcentration of gentamicin is 2 μg/mL. In some embodiments, thepredetermined concentration of gentamicin is 4 μg/mL.

Ciprofloxacin

In some embodiments, the at least one antimicrobial agent includesciprofloxacin. In some embodiments, the predetermined concentration ofciprofloxacin is greater than the CLSI MIC susceptible breakpoint. Insome embodiments, the supratherapeutic concentration of ciprofloxacin isgreater than the CLSI MIC intermediate breakpoint. In some embodiments,the predetermined concentration of ciprofloxacin is equal to the CLSIMIC resistant breakpoint.

In some embodiments, the predetermined concentration of ciprofloxacin isat least 4 μg/mL. In some embodiments, the predetermined concentrationof ciprofloxacin is 4 μg/mL.

Cefazolin

In some embodiments, the at least one antimicrobial agent includescefazolin. In some embodiments, the predetermined concentration ofcefazolin is greater than the CLSI MIC susceptible breakpoint. In someembodiments, the predetermined concentration of cefazolin is greaterthan the CLSI MIC intermediate breakpoint. In some embodiments, thepredetermined concentration of cefazolin is greater than the CLSI MICresistant breakpoint.

In some embodiments, the predetermined concentration of cefazolin isgreater than 40 μg/mL. In some embodiments, the predeterminedconcentration of cefazolin is greater than 50 μg/mL. In someembodiments, the predetermined concentration of cefazolin is at least 64μg/mL. In some embodiments, the predetermined concentration of cefazolinis 64 μg/mL.

Ceftriaxone

In some embodiments, the at least one antimicrobial agent includesceftriaxone. In some embodiments, the predetermined concentration ofceftriaxone is greater than the CLSI MIC susceptible breakpoint. In someembodiments, the predetermined concentration of ceftriaxone is greaterthan the CLSI MIC intermediate breakpoint. In some embodiments, thepredetermined concentration of ceftriaxone is greater than the CLSI MICresistant breakpoint.

In some embodiments, the predetermined concentration of ceftriaxone isgreater than 20 μg/mL. In some embodiments, the predeterminedconcentration of ceftriaxone is greater than 25 μg/mL. In someembodiments, the predetermined concentration of ceftriaxone is at least32 μg/mL. In some embodiments, the predetermined concentration ofceftriaxone is 32 μg/mL.

Cefepime

In some embodiments, the at least one antimicrobial agent includescefepime. In some embodiments, the predetermined concentration ofcefepime is greater than the CLSI MIC susceptible breakpoint. In someembodiments, the predetermined concentration of cefepime is greater thanthe CLSI MIC intermediate breakpoint. In some embodiments, thesupratherapeutic concentration of cefepime is greater than the CLSI MICresistant breakpoint.

In some embodiments, the predetermined concentration of cefepime isgreater than 30 μg/mL. In some embodiments, the predeterminedconcentration of cefepime is at least 32 μg/mL. In some embodiments, thepredetermined concentration of cefepime is greater than 40 μg/mL. Insome embodiments, the predetermined concentration of cefepime is greaterthan 50 μg/mL. In some embodiments, the predetermined concentration ofcefepime is at least 64 μg/mL. In some embodiments, the predeterminedconcentration of cefepime is 32 μg/mL. In some embodiments, thepredetermined concentration of cefepime is 64 μg/mL.

Ampicillin

In some embodiments, the at least one antimicrobial agent includesampicillin. In some embodiments, the predetermined concentration ofampicillin is greater than the CLSI MIC susceptible breakpoint. In someembodiments, the predetermined concentration of ampicillin is greaterthan the CLSI MIC intermediate breakpoint. In some embodiments, thepredetermined concentration of ampicillin is greater than the CLSI MICresistant breakpoint.

In some embodiments, the predetermined concentration of ampicillin isgreater than 50 μg/mL. In some embodiments, the predeterminedconcentration of ampicillin is greater than 100 μg/mL. In someembodiments, the predetermined concentration of ampicillin is at least128 μg/mL. In some embodiments, the predetermined concentration ofampicillin is 128 μg/mL.

Imipenem

In some embodiments, the at least one antimicrobial agent includesimipenem. In some embodiments, the predetermined concentration ofimipenem is greater than the CLSI MIC susceptible breakpoint. In someembodiments, the predetermined concentration of imipenem is greater thanthe CLSI MIC intermediate breakpoint. In some embodiments, thepredetermined concentration of imipenem is between the CLSI MICintermediate and resistant breakpoints. In some embodiments, thepredetermined concentration of imipenem is greater than the CLSI MICresistant breakpoint.

In some embodiments, the predetermined concentration of imipenem isgreater than 2 μg/mL. In some embodiments, the predeterminedconcentration of imipenem is greater than 3 μg/mL. In some embodiments,the predetermined concentration of imipenem is at least 4 μg/mL.

Trimethoprim

In some embodiments, the at least one antimicrobial agent includestrimethoprim. In some embodiments, the predetermined concentration oftrimethoprim is greater than the CLSI MIC susceptible breakpoint. Insome embodiments, the predetermined concentration of trimethoprim isgreater than the CLSI MIC intermediate breakpoint. In some embodiments,the predetermined concentration of trimethoprim is equal to theresistant breakpoint. In some embodiments, the predeterminedconcentration of trimethoprim is greater than the CLSI MIC resistantbreakpoint.

In some embodiments, the predetermined concentration of trimethoprim isat least 4 μg/mL. In some embodiments, the predetermined concentrationof trimethoprim is 4 μg/mL.

Sulfamethoxazole

In some embodiments, the at least one antimicrobial agent includessulfamethoxazole. In some embodiments, the predetermined concentrationof sulfamethoxazole is greater than the CLSI MIC susceptible breakpoint.In some embodiments, the predetermined concentration of sulfamethoxazoleis greater than the CLSI MIC intermediate breakpoint. In someembodiments, the predetermined concentration of sulfamethoxazole isequal to the CLSI MIC resistant breakpoint. In some embodiments, thepredetermined concentration of sulfamethoxazole is greater than the CLSIMIC resistant breakpoint.

In some embodiments, the predetermined concentration of sulfamethoxazoleis at least 76 μg/mL. In some embodiments, the predeterminedconcentration of sulfamethoxazole is 76 μg/mL

Amikacin

In some embodiments, the at least one antimicrobial agent includesamikacin. In some embodiments, the predetermined concentration ofampicillin is less than the CLSI MIC susceptible breakpoint. In someembodiments, the predetermined concentration of ampicillin is equal tothe CLSI MIC susceptible breakpoint. In some embodiments, thepredetermined concentration of ampicillin is greater than the CLSI MICsusceptible breakpoint. In some embodiments, the predeterminedconcentration of ampicillin is equal to the CLSI MIC intermediatebreakpoint.

In some embodiments, the predetermined concentration of amikacin is atleast 8 μg/mL. In some embodiments, the predetermined concentration ofamikacin is at least 16 μg/mL. In some embodiments, the predeterminedconcentration of amikacin is at least 32 μg/mL. In some embodiments, thepredetermined concentration of amikacin is 8 μg/mL. In some embodiments,the predetermined concentration of amikacin is 16 μg/mL. In someembodiments, the predetermined concentration of amikacin is 32 μg/mL.

Nitrofurantoin

In some embodiments, wherein the at least one antimicrobial agentincludes nitrofurantoin. In some embodiments, the predeterminedconcentration of nitrofurantoin is less than the CLSI MIC susceptiblebreakpoint.

In some embodiments, the predetermined concentration of nitrofurantoinis at least 16 μg/mL. In some embodiments, the predeterminedconcentration of nitrofurantoin is 6 μg/mL.

Fosfomycin

In some embodiments, wherein the at least one antimicrobial agentincludes fosfomycin. In some embodiments, the predeterminedconcentration of fosfomycin is less than the CLSI MIC susceptiblebreakpoint. In some embodiments, the predetermined concentration offosfomycin is equal to the CLSI MIC susceptible breakpoint.

In some embodiments, the predetermined concentration of fosfomycin is atleast 64 μg/mL. In some embodiments, the predetermined concentration offosfomycin is 64 μg/mL.

Piperacillin

In some embodiments, the at least one antimicrobial agent includespiperacillin. In some embodiments, the predetermined concentration ofpiperacillin is greater than the CLSI MIC susceptible breakpoint. Insome embodiments, the predetermined concentration of piperacillin isgreater than the CLSI MIC intermediate breakpoint. In some embodiments,the predetermined concentration of piperacillin is between the CLSI MICintermediate and resistant breakpoints. In some embodiments, thepredetermined concentration of piperacillin is greater than the CLSI MICresistant breakpoint.

In some embodiments, the predetermined concentration of piperacillin isgreater than 10 μg/mL. In some embodiments, the predeterminedconcentration of piperacillin is greater than 12 μg/mL. In someembodiments, the predetermined concentration of piperacillin is at least16 μg/mL.

Tazobactam

In some embodiments, the at least one antimicrobial agent includestazobactam. In some embodiments, the predetermined concentration oftazobactam is greater than the CLSI MIC susceptible breakpoint. In someembodiments, the predetermined concentration of tazobactam is greaterthan the CLSI MIC intermediate breakpoint. In some embodiments, thepredetermined concentration of tazobactam is between the CLSI MICintermediate and resistant breakpoints. In some embodiments, thepredetermined concentration of tazobactam is greater than the CLSI MICresistant breakpoint.

In some embodiments, the predetermined concentration of tazobactam isgreater than 2 μg/mL. In some embodiments, the predeterminedconcentration of tazobactam is greater than 3 μg/mL. In someembodiments, the predetermined concentration of tazobactam is at least 4μg/mL. In some configurations, Tazobactam can be used in combinationwith piperacillin in a combination antibiotic dosage for an AST test.Surprisingly, it was discovered that when varying the concentration ofthe combination dosage that acceptable results were found when theconcentration of Tazobactam was held constant and the dosages ofpiperacillin were varied in the following ratios 128/4, 64/4 and 32/4(piperacillin/tazobactam concentration in 3 μg/mL).

Amoxicillin

In some embodiments, the at least one antimicrobial agent includesamoxicillin. In some embodiments, the predetermined concentration ofamoxicillin is greater than the CLSI MIC susceptible breakpoint. In someembodiments, the predetermined concentration of amoxicillin is equal tothe CLSI MIC intermediate breakpoint. In some embodiments, thepredetermined concentration of amoxicillin is greater than the CLSI MICintermediate breakpoint. In some embodiments, the predeterminedconcentration of amoxicillin is equal to the CLSI MIC resistantbreakpoint. In some embodiments, the predetermined concentration ofamoxicillin is greater than the CLSI MIC resistant breakpoint.

In some embodiments, the predetermined concentration of amoxicillin isgreater than 16 μg/mL. In some embodiments, the predeterminedconcentration of amoxicillin is greater than 32 μg/mL. In someembodiments, the predetermined concentration of amoxicillin is at least16 μg/mL. In some embodiments, the predetermined concentration ofamoxicillin is at least 32 μg/mL. In some embodiments, the predeterminedconcentration of amoxicillin is at least 64 μg/mL. In some embodiments,the predetermined concentration of amoxicillin is 16 μg/mL. In someembodiments, the predetermined concentration of amoxicillin is 32 μg/mL.In some embodiments, the predetermined concentration of amoxicillin is64 μg/mL.

Clavulanate

In some embodiments, the at least one antimicrobial agent includesclavulanate. In some embodiments, the predetermined concentration ofclavulanate is greater than the CLSI MIC susceptible breakpoint. In someembodiments, the predetermined concentration of clavulanate is equal tothe CLSI MIC intermediate breakpoint. In some embodiments, thepredetermined concentration of clavulanate is greater than the CLSI MICintermediate breakpoint. In some embodiments, the predeterminedconcentration of clavulanate is equal to the CLSI MIC resistantbreakpoint. In some embodiments, the predetermined concentration ofclavulanate is greater than the CLSI MIC resistant breakpoint.

In some embodiments, the predetermined concentration of clavulanate isgreater than 8 μg/mL. In some embodiments, the predeterminedconcentration of clavulanate is greater than 16 μg/mL. In someembodiments, the predetermined concentration of clavulanate is at least8 μg/mL. In some embodiments, the predetermined concentration ofclavulanate is at least 16 μg/mL. In some embodiments, the predeterminedconcentration of clavulanate is at least 32 μg/mL. In some embodiments,the predetermined concentration of clavulanate is 8 μg/mL. In someembodiments, the predetermined concentration of clavulanate is 16 μg/mL.In some embodiments, the predetermined concentration of clavulanate is32 μg/mL.

Ertapenem

In some embodiments, the at least one antimicrobial agent includesertapenem. In some embodiments, the predetermined concentration ofertapenem is greater than the CLSI MIC susceptible breakpoint. In someembodiments, the predetermined concentration of ertapenem is equal tothe CLSI MIC intermediate breakpoint. In some embodiments, thepredetermined concentration of ertapenem is greater than the CLSI MICintermediate breakpoint. In some embodiments, the predeterminedconcentration of ertapenem is equal to the CLSI MIC resistantbreakpoint.

In some embodiments, the predetermined concentration of ertapenem isgreater than 2 μg/mL. In some embodiments, the predeterminedconcentration of ertapenem is greater than 4 μg/mL. In some embodiments,the predetermined concentration of ertapenem is at least 2 μg/mL. Insome embodiments, the predetermined concentration of ertapenem is atleast 4 μg/mL. In some embodiments, the predetermined concentration ofertapenem is 2 μg/mL. In some embodiments, the predeterminedconcentration of ertapenem is 4 μg/mL.

Meropenem

In some embodiments, the at least one antimicrobial agent includesmeropenem. In some embodiments, the predetermined concentration ofmeropenem is greater than the CLSI MIC susceptible breakpoint. In someembodiments, the predetermined concentration of meropenem is equal tothe CLSI MIC intermediate breakpoint. In some embodiments, thepredetermined concentration of meropenem is greater than the CLSI MICintermediate breakpoint. In some embodiments, the predeterminedconcentration of meropenem is equal to the CLSI MIC resistantbreakpoint.

In some embodiments, the predetermined concentration of meropenem isgreater than 2 μg/mL. In some embodiments, the predeterminedconcentration of meropenem is greater than 4 μg/mL. In some embodiments,the predetermined concentration of meropenem is at least 2 μg/mL. Insome embodiments, the predetermined concentration of meropenem is atleast 4 μg/mL. In some embodiments, the predetermined concentration ofmeropenem is 2 μg/mL. In some embodiments, the predeterminedconcentration of meropenem is 4 μg/mL.

In some embodiments, a microorganism is exposed to two or moreantimicrobial agents simultaneously. For instance, a culture media of aninoculate may comprise two or more antimicrobial agents. In someembodiments, a culture may comprise a beta-lactam antibiotic and abeta-lactamase inhibitor (BLI). In some embodiments, a culture mediacomprises two or more antimicrobial agents, wherein the two or moreantimicrobial agents are selected from the group of gentamicin,ciprofloxacin, cefazolin, ceftriaxone, cefepime, ampicillin,trimethoprim, sulfamethoxazole, amikacin, nitrofurantoin, fosfomycin,amoxicillin, clavulanate, ertapenem, and meropenem. In some embodiments,a culture media comprises trimethoprim and sulfamethoxazole. In someembodiments, a culture media comprises amoxicillin and clavulanate.

For some antimicrobial agents, and for some purposes, the predeterminedconcentration of the antimicrobial agent is at least 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95% or 100% or greater than the therapeutic concentration of theantimicrobial agent. In some embodiments, the predeterminedconcentration of the antimicrobial agent is at least 20% or greater thanthe therapeutic concentration of the antimicrobial agent. In someembodiments, the predetermined concentration of the antimicrobial agentis at least 40% or greater than the therapeutic concentration of theantimicrobial agent. In some embodiments, the predeterminedconcentration of the antimicrobial agent is at least 50% or greater thanthe therapeutic concentration of the antimicrobial agent. In someembodiments, the predetermined concentration of the antimicrobial agentis at least 70% or greater than the therapeutic concentration of theantimicrobial agent. In some embodiments, the predeterminedconcentration of the antimicrobial agent is at least 80% or greater thanthe therapeutic concentration of the antimicrobial agent. In someembodiments, a predetermined concentration of the antimicrobial agent isequal to the therapeutic concentration of the antimicrobial agent. Insome embodiments, the predetermined enhanced-rate concentration of theantimicrobial agent is less than the therapeutic concentration of theantimicrobial agent.

In some embodiments, the predetermined concentration of an antimicrobialagent is at least 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5,8, 8.5, 9, 9.5, or 10-fold or greater than the therapeutic concentrationof the antimicrobial agent. In some embodiments, the predeterminedenhanced-rate concentration of the antimicrobial agent is at least1.5-fold or greater than the therapeutic concentration of theantimicrobial agent. In some embodiments, the predeterminedenhanced-rate concentration of the antimicrobial agent is at least2-fold or greater than the predetermined enhanced-rate concentration ofthe antimicrobial agent. In some embodiments, the predeterminedenhanced-rate concentration of the antimicrobial agent is at least3-fold or greater than the therapeutic concentration of theantimicrobial agent. In some embodiments, the predeterminedenhanced-rate concentration of the antimicrobial agent is at least4-fold or greater than the therapeutic concentration of theantimicrobial agent. In some embodiments, the predeterminedenhanced-rate concentration of the antimicrobial agent is at least5-fold or greater than the therapeutic concentration of theantimicrobial agent.

In some embodiments, a predetermined concentration of an antimicrobialagent is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% or greater than thesusceptible CLSI MIC breakpoint of the antimicrobial agent. In someembodiments, a predetermined enhanced-rate concentration of theantimicrobial agent is at least 20% or greater than the susceptible CLSIMIC breakpoint of the antimicrobial agent. In some embodiments, apredetermined enhanced-rate concentration of the antimicrobial agent isat least 40% or greater than the susceptible CLSI MIC breakpoint of theantimicrobial agent. In some embodiments, a predetermined enhanced-rateconcentration of the antimicrobial agent is at least 50% or greater thanthe susceptible CLSI MIC breakpoint of the antimicrobial agent. In someembodiments, a predetermined enhanced-rate concentration of theantimicrobial agent is at least 70% or greater than the susceptible CLSIMIC breakpoint of the antimicrobial agent. In some embodiments, apredetermined enhanced-rate concentration of the antimicrobial agent isat least 80% or greater than the susceptible CLSI MIC breakpoint of theantimicrobial agent. In some embodiments, the predeterminedconcentration of an antimicrobial agent is equal to the susceptible CLSIMIC breakpoint. In some embodiments, the predetermined concentration ofan antimicrobial agent is less than the susceptible CLSI MIC breakpoint.

In some embodiments, the supratherapeutic concentration of anantimicrobial agent is at least 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6,6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10-fold or greater than the susceptibleCLSI MIC breakpoint of the antimicrobial agent. In some embodiments, thepredetermined enhanced-rate concentration of the antimicrobial agent isat least 1.5-fold or greater than the susceptible CLSI MIC breakpoint ofthe antimicrobial agent. In some embodiments, the predeterminedenhanced-rate concentration of the antimicrobial agent is at least2-fold or greater than the susceptible CLSI MIC breakpoint of theantimicrobial agent. In some embodiments, the predeterminedenhanced-rate concentration of the antimicrobial agent is at least3-fold or greater than the susceptible CLSI MIC breakpoint of theantimicrobial agent. In some embodiments, the predeterminedenhanced-rate concentration of the antimicrobial agent is at least4-fold or greater than the susceptible CLSI MIC breakpoint of theantimicrobial agent. In some embodiments, the predeterminedenhanced-rate concentration of the antimicrobial agent is at least5-fold or greater than the susceptible CLSI MIC breakpoint of theantimicrobial agent.

In some embodiments, a predetermined concentration of an antimicrobialagent is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% or greater than theintermediate CLSI MIC breakpoint of the antimicrobial agent. In someembodiments, a predetermined enhanced-rate concentration of theantimicrobial agent is at least 20% or greater than the intermediateCLSI MIC breakpoint of the antimicrobial agent. In some embodiments, apredetermined enhanced-rate concentration of the antimicrobial agent isat least 40% or greater than the intermediate CLSI MIC breakpoint of theantimicrobial agent. In some embodiments, a predetermined enhanced-rateconcentration of the antimicrobial agent is at least 50% or greater thanthe intermediate CLSI MIC breakpoint of the antimicrobial agent. In someembodiments, a predetermined enhanced-rate concentration of theantimicrobial agent is at least 70% or greater than the intermediateCLSI MIC breakpoint of the antimicrobial agent. In some embodiments, apredetermined enhanced-rate concentration of the antimicrobial agent isat least 80% or greater than the intermediate CLSI MIC breakpoint of theantimicrobial agent. In some embodiments, the predeterminedconcentration of an antimicrobial agent is equal to the intermediateCLSI MIC breakpoint. In some embodiments, the predeterminedconcentration of an antimicrobial agent is less than the intermediateCLSI MIC breakpoint.

In some embodiments, the supratherapeutic concentration of anantimicrobial agent is at least 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6,6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10-fold or greater than the intermediateCLSI MIC breakpoint of the antimicrobial agent. In some embodiments, thepredetermined enhanced-rate concentration of the antimicrobial agent isat least 1.5-fold or greater than the intermediate CLSI MIC breakpointof the antimicrobial agent. In some embodiments, the predeterminedenhanced-rate concentration of the antimicrobial agent is at least2-fold or greater than the intermediate CLSI MIC breakpoint of theantimicrobial agent. In some embodiments, the predeterminedenhanced-rate concentration of the antimicrobial agent is at least3-fold or greater than the intermediate CLSI MIC breakpoint of theantimicrobial agent. In some embodiments, the predeterminedenhanced-rate concentration of the antimicrobial agent is at least4-fold or greater than the intermediate CLSI MIC breakpoint of theantimicrobial agent. In some embodiments, the predeterminedenhanced-rate concentration of the antimicrobial agent is at least5-fold or greater than the intermediate CLSI MIC breakpoint of theantimicrobial agent.

In some embodiments, a predetermined concentration of an antimicrobialagent is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% or greater than theresistant CLSI MIC breakpoint of the antimicrobial agent. In someembodiments, a predetermined enhanced-rate concentration of theantimicrobial agent is at least 20% or greater than the resistant CLSIMIC breakpoint of the antimicrobial agent. In some embodiments, apredetermined enhanced-rate concentration of the antimicrobial agent isat least 40% or greater than the resistant CLSI MIC breakpoint of theantimicrobial agent. In some embodiments, a predetermined enhanced-rateconcentration of the antimicrobial agent is at least 50% or greater thanthe resistant CLSI MIC breakpoint of the antimicrobial agent. In someembodiments, a predetermined enhanced-rate concentration of theantimicrobial agent is at least 70% or greater than the resistant CLSIMIC breakpoint of the antimicrobial agent. In some embodiments, apredetermined enhanced-rate concentration of the antimicrobial agent isat least 80% or greater than the resistant CLSI MIC breakpoint of theantimicrobial agent. In some embodiments, the predeterminedconcentration of an antimicrobial agent is equal to the resistant CLSIMIC breakpoint. In some embodiments, the predetermined concentration ofan antimicrobial agent is less than the resistant CLSI MIC breakpoint.

In some embodiments, the supratherapeutic concentration of anantimicrobial agent is at least 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6,6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10-fold or greater than the resistantCLSI MIC breakpoint of the antimicrobial agent. In some embodiments, thepredetermined enhanced-rate concentration of the antimicrobial agent isat least 1.5-fold or greater than the resistant CLSI MIC breakpoint ofthe antimicrobial agent. In some embodiments, the predeterminedenhanced-rate concentration of the antimicrobial agent is at least2-fold or greater than the resistant CLSI MIC breakpoint of theantimicrobial agent. In some embodiments, the predeterminedenhanced-rate concentration of the antimicrobial agent is at least3-fold or greater than the resistant CLSI MIC breakpoint of theantimicrobial agent. In some embodiments, the predeterminedenhanced-rate concentration of the antimicrobial agent is at least4-fold or greater than the resistant CLSI MIC breakpoint of theantimicrobial agent. In some embodiments, the predeterminedenhanced-rate concentration of the antimicrobial agent is at least5-fold or greater than the resistant CLSI MIC breakpoint of theantimicrobial agent.

In some embodiments, the antimicrobial agent is an antibacterial agent.In some embodiments, the antibacterial agent is an antibiotic. In someembodiments, the antibiotic is a bactericidal antibiotic. In someembodiments, the antibiotic is a bacteriostatic antibiotic. In someembodiments, the antibiotic is selected from an aminoglycosideantibiotic, a beta-lactam antibiotic, an ansamycin antibiotic, amacrolide antibiotic, a sulfonamide antibiotic, a quinolone antibiotic,an oxazolidinone antibiotic, and a glycopeptide antibiotic.

In some embodiments, the antibiotic is a beta-lactam selected from2-(3-alanyl)clavam, 2-hydroxymethylclavam, 8-epi-thienamycin,acetyl-thienamycin, amoxicillin, amoxicillin sodium, amoxicillintrihydrate, amoxicillin-potassium clavulanate combination, ampicillin,ampicillin sodium, ampicillin trihydrate, ampicillin-sulbactam,apalcillin, aspoxicillin, azidocillin, azlocillin, aztreonam,bacampicillin, biapenem, carbenicillin, carbenicillin disodium,carfecillin, carindacillin, carpetimycin, cefacetril, cefaclor,cefadroxil, cefalexin, cefaloridine, cefalotin, cefamandole,cefamandole, cefapirin, cefatrizine, cefatrizine propylene glycol,cefazedone, cefazolin, cefbuperazone, cefcapene, cefcapene pivoxilhydrochloride, cefdinir, cefditoren, cefditoren pivoxil, cefepime,cefetamet, cefetamet pivoxil, cefixime, cefmenoxime, cefmetazole,cefminox, cefminox, cefmolexin, cefodizime, cefonicid, cefoperazone,ceforanide, cefoselis, cefotaxime, cefotetan, cefotiam, cefoxitin,cefozopran, cefpiramide, cefpirome, cefpodoxime, cefpodoxime proxetil,cefprozil, cefquinome, cefradine, cefroxadine, cefsulodin, ceftazidime,cefteram, cefteram pivoxil, ceftezole, ceftibuten, ceftizoxime,ceftriaxone, cefuroxime, cefuroxime axetil, cephalosporin, cephamycin,chitinovorin, ciclacillin, clavulanic acid, clometocillin, cloxacillin,cycloserine, deoxy pluracidomycin, dicloxacillin, dihydropluracidomycin, epicillin, epithienamycin, ertapenem, faropenem,flomoxef, flucloxacillin, hetacillin, imipenem, lenampicillin,loracarbef, mecillinam, meropenem, metampicillin, meticillin,mezlocillin, moxalactam, nafcillin, northienamycin, oxacillin,panipenem, penamecillin, penicillin, phenethicillin, piperacillin,tazobactam, pivampicillin, pivcefalexin, pivmecillinam, pivmecillinamhydrochloride, pluracidomycin, propicillin, sarmoxicillin, sulbactam,sulbenicillin, talampicillin, temocillin, terconazole, thienamycin, andticarcillin.

In some embodiments, the antibiotic is an aminoglycoside, selected from1,2′-N-DL-isoseryl-3′,4′-dideoxykanamycin B,1,2′-N-DL-isoseryl-kanamycin B,1,2′-N[(S)-4-amino-2-hydroxybutyryl]-3′,4′-dideoxykanamycin B,1,2′-N-[(S)-4-amino-2-hydroxybutyryq-kanamycin B,1-N-(2-Aminobutanesulfonyl) kanamycin A,1-N-(2-aminoethanesulfonyl)3,4′-dideoxyribostamycin,1-N-(2-Aminoethanesulfonyl)3′-deoxyribostamycin,1-N-(2-aminoethanesulfonyl)3′,4′-dideoxykanamycin B,1-N-(2-aminoethanesulfonyl)kanamycin A,1-N-(2-aminoethanesulfonyl)kanamycin B,1-N-(2-aminoethanesulfonyl)ribostamycin,1-N-(2-aminopropanesulfonyl)3′-deoxykanamycin B,1-N-(2-aminopropanesulfonyl)3′,4′-dideoxykanamycin B,1-N-(2-aminopropanesulfonyl)kanamycin A,1-N-(2-aminopropanesulfonyl)kanamycin B,1-N-(L-4-amino-2-hydroxy-butyryl)2,′3′-dideoxy-2′-fluorokanamycin A,1-N-(L-4-amino-2-hydroxy-propionyl)2,′3′-dideoxy-2′-fluorokanamycin A,1-N-DL-3′,4′-dideoxy-isoserylkanamycin B,1-N-DL-isoserylkanamycin,1-N-DL-isoserylkanamycin B,1-N[L+)-(alpha-hydroxy-gamma-aminobutyryl)]-XK-62-2,2′,3′-dideoxy-2′-fluorokanamycin A,2-hydroxygentamycin A3,2-hydroxygentamycin B, 2-hydroxygentamycin B1, 2-hydroxygentamycinJI-20A, 2-hydroxygentamycin JI-20B,3″-N-methyl-4″-C-methyl-3′,4′-dodeoxykanamycin A,3″-N-methyl-4″-C-methyl-3′,4′-dodeoxy kanamycin B,3″-N-methyl-4″-C-methyl-3′,4′-dodeoxy-6′-methyl kanamycin B,3′,4′-Dideoxy-3′-eno-ribostamycin,3′,4′-dideoxyneamine,3′,4′-dideoxyribostamycin,3′-deoxy-6′-N-methyl-kanamycin B,3′-deoxyneamine,3′-deoxyribostamycin,3′-oxysaccharocin,3,3′-nepotrehalosadiamine,3-demethoxy-2″-N-formimidoylistamycin B disulfate tetrahydrate,3-demethoxyistamycin B,3-O-demethyl-2-N-formimidoylistamycin B,3-O-demethylistamycin B,3-trehalosamine,4″, 6″-dideoxydibekacin,4-N-glycyl-KA-6606VI, 5″-Amino-3′,4′,5″-trideoxy-butirosin A,6″-deoxydibekacin,6′-epifortimicin A, 6-deoxy-neomycin (structure6-deoxy-neomycin B),6-deoxy-neomycin B, 6-deoxy-neomycin C,6-deoxy-paromomycin, acmimycin,AHB-3′,4′-dideoxyribostamycin,AHB-3′-deoxykanamycin B,AHB-3′-deoxyneamine,AHB-3′-deoxyribostamycin,AHB-4″-6″-dideoxydibekacin,AHB-6″-deoxydibekacin, AHB-dideoxyneamine,AHB-kanamycin B,AHB-methyl-3′-deoxykanamycin B, amikacin, amikacin sulfate, apramycin,arbekacin, astromicin, astromicin sulfate, bekanamycin, bluensomycin,boholmycin, butirosin, butirosin B, catenulin, coumamidine gammal,coumamidine gamma2,D,L-1-N-(alpha-hydroxy-beta-aminopropionyl)-XK-62-2,dactimicin,de-O-methyl-4-N-glycyl-KA-6606VI,de-O-methyl-KA-66061,de-O-methyl-KA-70381,destomycin A, destomycin B,di-N6′,03-demethylistamycin A, dibekacin, dibekacin sulfate,dihydrostreptomycin, dihydrostreptomycin sulfate,epi-formamidoylglycidylfortimicin B, epihygromycin,formimidoyl-istamycin A, formimidoyl-istamycin B, fortimicin B,fortimicin C, fortimicin D, fortimicin KE, fortimicin KF, fortimicin KG,fortimicin KG1 (stereoisomer KG1/KG2), fortimicin KG2(stereoisomerKG1/KG2), fortimicin KG3, framycetin, framycetin sulphate, gentamicin,gentamycin sulfate, globeomycin, hybrimycin A1, hybrimycin A2,hybrimycin B1, hybrimycin B2, hybrimycin C1, hybrimycin C2,hydroxystreptomycin, hygromycin, hygromycin B, isepamicin, isepamicinsulfate, istamycin, kanamycin, kanamycin sulphate, kasugamycin,lividomycin, marcomycin, micronomicin, micronomicin sulfate, mutamicin,myomycin, N-demethyl-7-O-demethylcelesticetin, demethylcelesticetin,methanesulfonic acid derivative of istamycin, nebramycin, nebramycin,neomycin, netilmicin, oligostatin, paromomycin, quintomycin,ribostamycin, saccharocin, seldomycin, sisomicin, sorbistin,spectinomycin, streptomycin, tobramycin, trehalosmaine, trestatin,validamycin, verdamycin, xylostasin, and zygomycin;

In some embodiments, the antibiotic is an ansa-type antibiotic selectedfrom 21-hydroxy-25-demethyl-25-methylthioprotostreptovaricin,3-methylthiorifamycin, ansamitocin, atropisostreptovaricin, awamycin,halomicin, maytansine, naphthomycin, rifabutin, rifamide, rifampicin,rifamycin, rifapentine, rifaximin, rubradirin, streptovaricin, andtolypomycin.

In some embodiments, the antibiotic is an anthraquinone selected fromauramycin, cinerubin, ditrisarubicin, ditrisarubicin C, figaroic acidfragilomycin, minomycin, rabelomycin, rudolfomycin, and sulfurmycin.

In some embodiments, the antibiotic is an azole selected fromazanidazole, bifonazole, butoconazol, chlormidazole, chlormidazolehydrochloride, cloconazole, cloconazole monohydrochloride, clotrimazol,dimetridazole, econazole, econazole nitrate, enilconazole,fenticonazole, fenticonazole nitrate, fezatione, fluconazole,flutrimazole, isoconazole, isoconazole nitrate, itraconazole,ketoconazole, lanoconazole, metronidazole, metronidazole benzoate,miconazole, miconazole nitrate, neticonazole, nimorazole, niridazole,omoconazol, ornidazole, oxiconazole, oxiconazole nitrate, propenidazole,secnidazol, sertaconazole, sertaconazole nitrate, sulconazole,sulconazole nitrate, tinidazole, tioconazole, and voriconazol.

In some embodiments, the antibiotic is a glycopeptide selected fromacanthomycin, actaplanin, avoparcin, balhimycin, bleomycin B (copperbleomycin), chloroorienticin, chloropolysporin, demethylvancomycin,enduracidin, galacardin, guanidylfungin, hachimycin, demethylvancomycin,N-nonanoyl-teicoplanin, phleomycin, platomycin, ristocetin,staphylocidin, talisomycin, teicoplanin, vancomycin, victomycin,xylocandin, and zorbamycin.

In some embodiments, the antibiotic is a macrolide selected fromacetylleucomycin, acetylkitasamycin, angolamycin, azithromycin,bafilomycin, brefeldin, carbomycin, chalcomycin, cirramycin,clarithromycin, concanamycin, deisovaleryl-niddamycin,demycinosyl-mycinamycin, Di-O-methyltiacumicidin, dirithromycin,erythromycin, erythromycin estolate, erythromycin ethyl succinate,erythromycin lactobionate, erythromycin stearate, flurithromycin,focusin, foromacidin, haterumalide, haterumalide, josamycin, josamycinropionate, juvenimycin, juvenimycin, kitasamycin, ketotiacumicin,lankavacidin, lankavamycin, leucomycin, machecin, maridomycin,megalomicin, methylleucomycin, methymycin, midecamycin, miocamycin,mycaminosyltylactone, mycinomycin, neutramycin, niddamycin, nonactin,oleandomycin, phenylacetyldeltamycin, pamamycin, picromycin,rokitamycin, rosaramicin, roxithromycin, sedecamycin, shincomycin,spiramycin, swalpamycin, tacrolimus, telithromycin, tiacumicin,tilmicosin, treponemycin, troleandomycin, tylosin, and venturicidin.

In some embodiments, the antibiotic is a nucleoside selected fromamicetin, angustmycin, azathymidine, blasticidin S, epiroprim,flucytosine, gougerotin, mildiomycin, nikkomycin, nucleocidin,oxanosine, oxanosine, puromycin, pyrazomycin, showdomycin, sinefungin,sparsogenin, spicamycin, tunicamycin, uracil polyoxin, and vengicide.

In some embodiments, the antibiotic is a peptide selected fromactinomycin, aculeacin, alazopeptin, amfomycin, amythiamycin, antifungalfrom Zalerion arboricola, antrimycin, apid, apidaecin, aspartocin,auromomycin, bacileucin, bacillomycin, bacillopeptin, bacitracin,bagacidin, berninamycin, beta-alanyl-L-tyrosine, bottromycin,capreomycin, caspofungine, cepacidine, cerexin, cilofungin, circulin,colistin, cyclodepsipeptide, cytophagin, dactinomycin, daptomycin,decapeptide, desoxymulundocandin, echanomycin, echinocandin B,echinomycin, ecomycin, enniatin, etamycin, fabatin, ferrimycin,ferrimycin, ficellomycin, fluoronocathiacin, fusaricidin, gardimycin,gatavalin, globopeptin, glyphomycin, gramicidin, herbicolin, iomycin,iturin, iyomycin, izupeptin, j aniemycin, j anthinocin, j olipeptin,katanosin, killertoxin, lipopeptide antibiotic, lipopeptide fromZalerion sp., lysobactin, lysozyme, macromomycin, magainin, melittin,mersacidin, mikamycin, mureidomycin, mycoplanecin, mycosubtilin,neopeptifluorin, neoviridogrisein, netropsin, nisin, nocathiacin,nocathiacin 6-deoxyglycoside, nosiheptide, octapeptin, pacidamycin,pentadecapeptide, peptifluorin, permetin, phytoactin, phytostreptin,planothiocin, plusbacin, polcillin, polymyxin antibiotic complex,polymyxin B, polymyxin B1, polymyxin F, preneocarzinostatin, quinomycin,quinupristin-dalfopristin, safracin, salmycin, salmycin, salmycin,sandramycin, saramycetin, siomycin, sperabillin, sporamycin, astreptomyces compound, subtilin, teicoplanin aglycone, telomycin,thermothiocin, thiopeptin, thiostrepton, tridecaptin, tsushimycin,tuberactinomycin, tuberactinomycin, tyrothricin, valinomycin, viomycin,virginiamycin, and zervacin.

In some embodiments, the antibiotic is a polyene selected fromamphotericin, amphotericin, aureofungin, ayfactin, azalomycin,blasticidin, candicidin, candicidin methyl ester, candimycin, candimycinmethyl ester, chinopricin, filipin, flavofungin, fradicin, hamycin,hydropricin, levorin, lucensomycin, lucknomycin, mediocidin, mediocidinmethyl ester, mepartricin, methylamphotericin, natamycin, niphimycin,nystatin, nystatin methyl ester, oxypricin, partricin, pentamycin,perimycin, pimaricin, primycin, proticin, rimocidin, sistomycosin,sorangicin, and trichomycin.

In some embodiments, the antibiotic is a polyether selected from20-deoxy-epi-narasin, 20-deoxysalinomycin, carriomycin, dianemycin,dihydrolonomycin, etheromycin, ionomycin, iso-lasalocid, lasalocid,lenoremycin, lonomycin, lysocellin, monensin, narasin, oxolonomycin, apolycyclic ether antibiotic, and salinomycin.

In some embodiments, the antibiotic is a quinolone selected fromalkyl-methylendioxy-4(1H)-oxocinnoline-3-carboxylic acid,alatrofloxacin, cinoxacin, ciprofloxacin, ciprofloxacin hydrochloride,danofloxacin, dermofongin A, enoxacin, enrofloxacin, fleroxacin,flumequine, gatifloxacin, gemifloxacin, grepafloxacin, levofloxacin,lomefloxacin, lomefloxacin, hydrochloride, miloxacin, moxifloxacin,nadifloxacin, nalidixic acid, nifuroquine, norfloxacin, ofloxacin,orbifloxacin, oxolinic acid, pazufloxacine, pefloxacin, pefloxacinmesylate, pipemidic acid, piromidic acid, premafloxacin, rosoxacin,rufloxacin, sparfloxacin, temafloxacin, tosufloxacin, and trovafloxacin.

In some embodiments, the antibiotic is a steroid selected fromaminosterol, ascosteroside, cladosporide, dihydrofusidic acid,dehydro-dihydrofusidic acid, dehydrofusidic acid, fusidic acid, andsqualamine.

In some embodiments, the antibiotic is a sulfonamide selected fromchloramine, dapsone, mafenide, phthalylsulfathiazole,succinylsulfathiazole, sulfabenzamide, sulfacetamide,sulfachlorpyridazine, sulfadiazine, sulfadiazine silver, sulfadicramide,sulfadimethoxine, sulfadoxine, sulfaguanidine, sulfalene, sulfamazone,sulfamerazine, sulfamethazine, sulfamethizole, sulfamethoxazole,sulfamethoxypyridazine, sulfamonomethoxine, sulfamoxol, sulfanilamide,sulfaperine, sulfaphenazol, sulfapyridine, sulfaquinoxaline,sulfasuccinamide, sulfathiazole, sulfathiourea, sulfatolamide,sulfatriazin, sulfisomidine, sulfisoxazole, sulfisoxazole acetyl, andsulfacarbamide.

In some embodiments, the antibiotic is a tetracycline selected fromdihydrosteffimycin, demethyltetracycline, aclacinomycin, akrobomycin,baumycin, bromotetracycline, cetocyclin, chlortetracycline,clomocycline, daunorubicin, demeclocycline, doxorubicin, doxorubicinhydrochloride, doxycycline, lymecyclin, marcellomycin, meclocycline,meclocycline sulfosalicylate, methacycline, minocycline, minocyclinehydrochloride, musettamycin, oxytetracycline, rhodirubin,rolitetracycline, rubomycin, serirubicin, steffimycin, and tetracycline.

In some embodiments, the antibiotic is a dicarboxylic acid selected fromadipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid,1,11-undecanedioic acid, 1,12-dodecanedioic acid, 1,13-tridecanedioicacid, and 1,14-tetradecanedioic acid.

In some embodiments, the antibiotic is an antibiotic metal or a metalion, wherein the metal is selected from silver, copper, zinc, mercury,tin, lead, bismutin, cadmium, chromium, and gold.

In some embodiments, the antibiotic is a silver compound selected fromsilver acetate, silver benzoate, silver carbonate, silver iodate, silveriodide, silver lactate, silver laurate, silver nitrate, silver oxide,silver palmitate, silver protein, and silver sulfadiazine.

In some embodiments, the antibiotic is an oxidizing agent or a substancethat releases free radicals or active oxygen, selected from oxygen,hydrogen peroxide, benzoyl peroxide, elemental halogen species,oxygenated halogen species, bleaching agents, perchlorite species,iodine, iodate, and benzoyl peroxide.

In some embodiments, the antibiotic is a cationic antimicrobial agentselected from quaternary ammonium compounds, alkyltrimethyl ammoniumbromide, cetrimide, benzalkonium chloride, n-alkyldimethylbenzylammonium chloride, dialkylmethyl ammonium halide, and dialkylbenzylammonium halide;

In some embodiments, the antibiotic is a compound selected fromchlorhexidine acetate, chlorhexidine gluconate and chlorhexidinehydrochloride, picloxydine, alexidine, polihexanide, chlorproguanilhydrochloride, proguanil hydrochloride, metformin hydrochloride,phenformin, and buformin hydrochloride.

In some embodiments, the antibiotic is an agent selected from abomycin,acetomycin, acetoxycycloheximide, acetylnanaomycin, an actinoplanessp.Compound, actinopyrone, aflastatin, albacarcin, albacarcin, albofungin,albofungin, alisamycin, alpha-R,S-methoxycarbonylbenzylmonate,altromycin, amicetin, amycin, amycin demanoyl compound, amycine,amycomycin, anandimycin, anisomycin, anthramycin, anti-syphilis imunesubstance, anti-tuberculosis immune substance, antibiotic fromEschericia coli, antibiotics from Streptomycesrefuineus, anticapsin,antimycin, aplasmomycin, aranorosin, aranorosinol, arugomycin,ascofuranone, ascomycin, ascosin, Aspergillus flavus antibiotic,asukamycin, aurantinin, an Aureolic acid antibiotic substance, aurodox,avilamycin, azidamfenicol, azidimycin, bacillaene, a Bacillus larvaeantibiotic, bactobolin, benanomycin, benzanthrin, benzylmonate,bicozamycin, bravomicin, brodimoprim, butalactin, calcimycin, calvaticacid, candiplanecin, carumonam, carzinophilin, celesticetin, cepacin,cerulenin, cervinomycin, chartreusin, chloramphenicol, chloramphenicolpalmitate, chloramphenicol succinate sodium, chlorflavonin,chlorobiocin, chlorocarcin, chromomycin, ciclopirox, ciclopirox olamine,citreamicin, cladosporin, clazamycin, clecarmycin, clindamycin,coliformin, collinomycin, copiamycin, corallopyronin, corynecandin,coumermycin, culpin, cuprimyxin, cyclamidomycin, cycloheximide,dactylomycin, danomycin, danubomycin, delaminomycin, demethoxyrapamycin,demethylscytophycin, dermadin, desdamethine, dexylosyl-benanomycin,pseudoaglycone, dihydromocimycin, dihydronancimycin, diumycin, dnacin,dorrigocin, dynemycin, dynemycin triacetate, ecteinascidin, efrotomycin,endomycin, ensanchomycin, equisetin, ericamycin, esperamicin,ethylmonate, everninomicin, feldamycin, flambamycin, flavensomycin,florfenicol, fluvomycin, fosfomycin, fosfonochlorin, fredericamycin,frenolicin, fumagillin, fumifungin, funginon, fusacandin, fusafungin,gelbecidine, glidobactin, grahamimycin, granaticin, griseofulvin,griseoviridin, grisonomycin, hayumicin, hayumicin, hazymicin, hedamycin,heneicomycin, heptelicid acid, holomycin, humidin, isohematinic acid,karnatakin, kazusamycin, kristenin, L-dihydrophenylalanine, aL-isoleucyl-L-2-amino-4-(4′-amino-2′, 5′-cyclohexadienyl) derivative,lanomycin, leinamycin, leptomycin, libanomycin, lincomycin, lomofungin,lysolipin, magnesidin, manumycin, melanomycin,methoxycarbonylmethylmonate, methoxycarbonylethylmonate,methoxycarbonylphenylmonate, methyl pseudomonate, methylmonate,microcin, mitomalcin, mocimycin, moenomycin, monoacetyl cladosporin,monomethyl cladosporin, mupirocin, mupirocin calcium, mycobacidin,myriocin, myxopyronin, pseudoaglycone, nanaomycin, nancimycin,nargenicin, neocarcinostatin, neoenactin, neothramycin, nifurtoinol,nocardicin, nogalamycin, novobiocin, octylmonate, olivomycin,orthosomycin, oudemansin, oxirapentyn, oxoglaucine methiodide, pactacin,pactamycin, papulacandin, paulomycin, phaeoramularia fungicide,phenelfamycin, phenyl, cerulenin, phenylmonate, pholipomycin,pirlimycin, pleuromutilin, a polylactone derivative, polynitroxin,polyoxin, porfiromycin, pradimicin, prenomycin, Prop-2-enylmonate,protomycin, Pseudomonas antibiotic, pseudomonic acid, purpuromycin,pyrinodemin, pyrrolnitrin, pyrrolomycin, amino, chloro pentenedioicacid, rapamycin, rebeccamycin, resistomycin, reuterin, reveromycin,rhizocticin, roridin, rubiflavin, naphthyridinomycin, saframycin,saphenamycin, sarkomycin, sarkomycin, sclopularin, selenomycin,siccanin, spartanamicin, spectinomycin, spongistatin, stravidin,streptolydigin, streptomycesarenae antibiotic complex, streptonigrin,streptothricins, streptovitacin, streptozotocine, a strobilurinderivative, stubomycin, sulfamethoxazol-trimethoprim, sakamycin,tejeramycin, terpentecin, tetrocarcin, thermorubin, thermozymocidin,thiamphenicol, thioaurin, thiolutin, thiomarinol, thiomarinol,tirandamycin, tolytoxin, trichodermin, trienomycin, trimethoprim,trioxacarcin, tyrissamycin, umbrinomycin, unphenelfamycin, urauchimycin,usnic acid, uredolysin, variotin, vermisporin, verrucarin, and analogs,salts and derivatives thereof.

In some embodiments, the antibiotic is selected from the group ofaminoglycoside, ansamycin, carbacephem, carbapenem, cephalosporin,fosfomycin, glycopeptide, lincosamide, lipopeptide, macrolide,monobactam, nitrofuran, oxazolidinone, penicillin, quinolone,sulfonamide, and tetracycline.

In some embodiments, at least 1, 2, 3, 4, or 5 or more antimicrobialagents are selected from the group of aminoglycoside, ansamycin,carbacephem, carbapenem, cephalosporin, fosfomycin, glycopeptide,lincosamide, lipopeptide, macrolide, monobactam, nitrofuran,oxazolidinone, penicillin, quinolone, sulfonamide, and tetracycline.

In some embodiments, at least one antimicrobial agent is cephalosporin.In some embodiments, the cephalosporin is selected from the group offirst generation cephalosporin, second generation cephalosporin, thirdgeneration cephalosporin, fourth generation cephalosporin, and fifthgeneration cephalosporin.

In some embodiments, at least one antimicrobial agent is quinolone. Insome embodiments, quinolone is a fluoroquinolone.

In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, or 15 or more antimicrobial agents are selected from the group ofgentamicin, ciprofloxacin, cefazolin, ceftriaxone, cefepime, ampicillin,imipenem, trimethoprim, sulfamethoxazole, amikacin, nitrofurantoin,fosfomycin, piperacillin, tazobactam, amoxicillin, and clavulanate.

In some embodiments, the antibiotic is selected from the group ofgentamicin, ciprofloxacin, cefazolin, ceftriaxone, cefepime, ampicillin,imipenem, trimethoprim, sulfamethoxazole, amikacin, nitrofurantoin,fosfomycin, piperacillin, tazobactam, amoxicillin, and clavulanate.

In some embodiments, the at least one antimicrobial agent includes abeta-lactamase inhibitor. In some embodiments, the beta-lactamaseinhibitor is selected from clavulanate, sulbactam, tazobactam,avibactam, relebactam, tebipenem, y-methylidene Penem, and boron basedtransition state inhibitors. In some embodiments, the beta-lactamaseinhibitor is accompanied by a beta-lactam antibiotic.

The disclosure illustratively described herein can suitably be practicedin the absence of any element or elements, limitation or limitations,not specifically disclosed herein. Thus, for example, the terms“comprising”, “including,” containing”, etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof, but it is recognized that various modifications arepossible within the scope of the disclosure claimed.

While this invention has been described with reference to illustrativeembodiments and examples, the description is not intended to beconstrued in a limiting sense. Thus, various modifications of theillustrative embodiments, as well as other embodiments of the invention,will be apparent to persons skilled in the art upon reference to thisdescription. It is therefore contemplated that the appended claims willcover any such modifications or embodiments.

All publications, patents and patent applications referred to herein areincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

What is claimed is:
 1. A method for determining the susceptibility ofbacteria in a clinical sample or an inoculant derived therefrom to anantibiotic agent, the method comprising: a) inoculating a test portionof the clinical sample in a medium containing a predeterminedrate-targeted concentration of the antibiotic agent; b) inoculating acontrol portion of the clinical sample in a medium that does not containthe antibiotic agent; c) incubating the test portion for an incubationperiod; d) incubating the control portion for the incubation period; e)determining a quantity of RNA in the test portion and a quantity of RNAin the control portion at the conclusion of the incubation period thatis less than 480 minutes after the completion of step a); and f)determining a susceptibility of the bacteria to the antibiotic agent bycomparing the quantity of RNA in the test portion to the quantity of theRNA in the control portion.
 2. The method of claim 1, wherein incubatingthe test portion is done within a test incubation chamber on acentrifugal disc, and incubating the control portion is done within acontrol incubation chamber on the same centrifugal disc.
 3. The methodof claim 2, wherein the test incubation chamber is fluidically isolatedfrom the control incubation chamber.
 4. The method of claim 1, whereinthe RNA comprises at least one of pre-ribosomal RNA, mature RNA,ribosomal RNA, 16S rRNA and 23S rRNA. 5.-8. (canceled)
 9. The method ofclaim 1, wherein the incubation period is equal to or less than 450minutes. 10.-23. (canceled)
 24. The method of claim 1, wherein theantibiotic agent comprises at least one of Gentamicin, Ciprofloxacin,Cefazolin, Ceftriaxone, Cefepime, Ampicillin,Trimethoprim-Sulfamethoxazole, Nitrofurantoin, Fosfomycin,Amoxicillin-Clavulanate, Amikacin, Ertapenem, Meropenem and combinationsthereof.
 25. (canceled)
 26. (canceled)
 27. The method of claim 1,wherein the predetermined rate-targeted concentration is equal to orabove the resistant CLSI MIC cutoff (for urine) for the antibioticagent.
 28. The method of claim 27, wherein the predeterminedrate-targeted concentration is at least 2-fold or greater than theresistant CLSI MIC cutoff (for urine) for the antibiotic agent. 29.-103.(canceled)
 104. The method of claim 1, wherein the bacteria is anunknown bacteria when steps a) to f) of claim 1 are conducted.
 105. Themethod of claim 1, further comprising lysing the test portion prior todetermining the quantity of RNA in the test portion.
 106. The method ofclaim 105, further comprising the steps of g) subjecting the testportion to mechanical lysis to cause disruption of a cellular membranein the bacteria; h) contacting the test portion with an alkalinematerial to produce a lysate composition comprising the RNA; and i)recovering the lysate composition from the test portion.
 107. The methodof claim 106, wherein Step h) comprises contacting the bacteria in thetest portion with an alkaline liquid. 108.-115. (canceled)
 116. Themethod of claim 105, wherein incubating the test portion is done withina test incubation chamber on a centrifugal disc, and lysing the testportion is conducted within a lysing chamber on the same centrifugaldisc. 117.-133. (canceled)
 134. The method of claim 105, wherein Step h)is carried out after commencement of disruption of the cellular membranein Step g).
 135. The method of claim 1, wherein the bacteria aresusceptible to the antibiotic agent if the quantity of RNA in thecontrol portion is more than the quantity of RNA in the test portion atthe conclusion of the incubation period.
 136. The method of claim 1,wherein the bacteria are not susceptible to the antibiotic agent if thequantity of RNA in the control portion is nearly equal, equal, or lessthan the quantity of RNA in the test portion at the conclusion of theincubation period.
 137. The method of claim 1, wherein the microorganismis susceptible to the antibiotic agent when the quantity of RNA in thetest portion is about 40% or less of the quantity of RNA in the controlportion at the conclusion of the incubation period. 138.-430. (canceled)431. The method of claim 1, wherein the clinical sample comprisesmammalian cellular material.
 432. The method of claim 1, wherein thesample comprises a bodily fluid selected from the group consisting ofblood, urine, saliva, sweat, tears, mucus, breast milk, plasma, serum,synovial fluid, pleural fluid, lymph fluid, amniotic fluid, feces,cerebrospinal fluid, and any mixture of two or more of these.
 433. Themethod of claim 432, wherein the sample comprises an inoculant derivedfrom the bodily fluid.